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294 p.- 19 fig., INTRODUCTION Microspore embryogenesis represents a unique system of single cell reprogramming in plants in which a highly specialized cell, the microspore, by specific stress treatment, switches its fate towards an embryogenesis pathway forming haploid embryos and producing haploid and doubled haploid plants. This process is a potent biotechnological tool for obtaining doubled haploids in many crops to be used in plant breeding programs. The application of this process has important limitations because the mechanisms of microspore embryogenesis induction and progression, which involve proliferation and differentiation events, are not yet completely elucidated. The monocot Hordeum vulgare (barley) and the dicot Brassica napus (rapeseed) are model systems for the process in which direct embryogenesis is induced, via different temperature stress treatments, in isolated microspores cultured in liquid media., OBJECTIVES AND RESULTS In this PhD thesis, isolated microspore cultures were induced by different temperatures in two species, by 32ºC in rapeseed, and by 4ºC in barley. Previous studies of our group suggested some factors that would be involved in the microspore embryogenesis initiation and progression. Three of these factors have been analysed in the two species by a multidisciplinary approach: 1) DNA methylation, 2) Arabinogalactan proteins (AGPs) and 3) Endogenous auxin. The results were compared in the two species in order to characterize common mechanisms involved in microspore embryogenesis in monocot and dicot species. We studied their dynamics during the process of microspore embryogenesis, as well as their function by analysing the effect of inhibitors for these factors on the embryogenesis induction and efficiency. In addition, some of them were also analysed during pollen development to identify changes associated with the change of developmental program towards microspore embryogenesis. The results indicated that these factors can be used as early markers of microspore embryogenesis. Since they are found in the two species analysed, independently of the inductor stress, they would inform on general mechanisms involved in the process that could be extended to other plant species. In this thesis, the dynamics of global DNA methylation was studied during microspore embryogenesis and gametophytic development in Hordeum vulgare, and the results were compared with those recently reported by our group in B. napus. The quantifications of global DNA methylation levels and the 5-methyl-deoxycytidine (5mdC) immunofluorescence assays showed low levels of DNA methylation in microspores and a high increase along gametophytic development and pollen maturation. The 5mdC signal increased in the generative and sperm nuclei whereas the vegetative nucleus showed lower signal. After cold stress and microspore embryogenesis induction, low levels of DNA methylation and weak signal of 5mdC were observed in nuclei of responsive microspores and 2 – 4 cell proembryos, indicating that a global hypomethylation occurs during the change of the developmental program and first embryogenic divisions in barley, as it was reported in rapeseed. At later developmental stages, the global DNA methylation was high in developing embryos and it increased during embryo maturation suggesting that DNA methylation is critical for embryo formation and plays a role in the regulation of gene expression in microspore embryogenesis., This thesis also analysed the effects of 5-azacytidine (AzaC), a DNA methylation inhibitor, on microspore embryogenesis initiation and progression in the two species, rapeseed and barley. AzaC treatments decreased global DNA methylation levels and favoured the initiation of microspore embryogenesis, increasing the production of multicellular embryos compared with untreated cultures. In contrast, this agent impaired the progression of microspore embryogenesis, at later stages. Taken together, the results indicate that DNA demethylation by AzaC promotes microspore embryogenesis initiation, while embryo differentiation requires de novo DNA methylation and is prevented by AzaC., The dynamics of the presence and distribution of arabinogalactan proteins (AGPs) were studied during microspore embryogenesis in Brassica napus and Hordeum vulgare, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM 14, JIM 13 and MAC 207). The results showed that AGPs were induced during microspore embryogenesis and the blocking of AGPs with Yariv reagent impaired the process, in both species. This indicates that AGPs play a key role in microspore embryo development. AGPs epitopes were localized on cell walls and cytoplasmic spots suggesting an active production and secretion of AGPs during microspore reprogramming and embryo development. The distribution patterns of AGPs were also analysed during pollen development in Brassica napus and the results indicated that several AGPs are also related to pollen maturation and germination. It was also analysed the expression pattern of the BnAGP Sta – 4 gene in B. napus. The results showed an increase in the expression of the Sta 39 – 4 gene after microspore embryogenesis initiation suggesting that AGPs are induced with the switch of developmental program, constituting early markers of microspore embryogenesis. These results provide new evidence of the role of endogenous AGPs as potential regulating molecules of the process. In this thesis, the dynamics of endogenous auxin was studied during the microspore reprogramming and embryogenesis in the monocot H. vulgare, and the results were compared with those recently reported by our group in B. napus. The results obtained in this thesis provide information about the changes of auxin distribution during microspore embryogenesis in barley, a model system of monocot species., The results showed that auxin increased during early stages of microspore embryogenesis, findings that are consistent with previous results of our group on auxin distribution in microspore embryogenesis of Brassica napus and Quercus suber. It was also analysed, in the present work, the effect of N-1-naphthylphthalamic acid (NPA), inhibitor of polar auxin transport, and P-chlorophenoxyisobutytric acid (PCIB) auxin action inhibitor, on microspore embryogenesis. The analysis revealed that NPA treatment decreased the number of multicellular embryos, after the induction by stress, compared with untreated cultures; PCIB treatments completely inhibited the responsive microspores to reprogram to embryogenesis. These results indicate that polar auxin transport and auxin action have a key role in initiation and progression of microspore embryogenesis in barley, as it was reported in rapeseed, suggesting a common general role of auxin in the process., CONCLUSION In conclusion, the results in this PhD thesis revealed the involvement of DNA methylation, AGPs and auxin in microspore embryogenesis initiation and progression, possibly with common roles in dicot and monocot species. The results suggest that the induction of microspore embryogenesis requires DNA hypomethylation, AGPs induction and auxin accumulation, independently of the inductor stress and the species. Our results could open a way to design new biotechnological strategies for improving doubled-haploid production in breeding programs of horticulture and cereal crops., Beca del Programa JAE Predoctoral del CSIC (JAEPre 010-052). - Proyectos del Plan Nacional: BFU2008-00203 ; BFU2011-23752 ; AGL2014 – 52028-R -Proyecto de cooperación internacional: Acción integrada Hispano-Portuguesa PRI-AIBPT-2011-0763, El trabajo de esta tesis ha dado lugar a tres publicaciones en revistas JCR (archivos permitidos depositados en Digital.CSIC) y un manuscrito en preparación. 1) Change in DNA methylation levels and nuclear distribution patterns after microspore reprogramming to embryogenesis in barley. http://digital.csic.es/handle/10261/101305 2) 5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley. https://digital.csic.es/handle/10261/118199 3) Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus. https://digital.csic.es/handle/10261/89731. Sin archivo, ver http://dx.doi.org/10.1007/s00497-013-0217-8 4) Auxin increase and distribution, and effects of auxin inhibitors on microspore embryogenesis initiation and progression in barley. (En preparación).

294 p.- 19 fig. INTRODUCTION Microspore embryogenesis represents a unique system of single cell reprogramming in plants in which a highly specialized cell, the microspore, by specific stress treatment, switches its fate towards an embryogenesis pathway forming haploid embryos and producing haploid and doubled haploid plants. This process is a potent biotechnological tool for obtaining doubled haploids in many crops to be used in plant breeding programs. The application of this process has important limitations because the mechanisms of microspore embryogenesis induction and progression, which involve proliferation and differentiation events, are not yet completely elucidated. The monocot Hordeum vulgare (barley) and the dicot Brassica napus (rapeseed) are model systems for the process in which direct embryogenesis is induced, via different temperature stress treatments, in isolated microspores cultured in liquid media. OBJECTIVES AND RESULTS In this PhD thesis, isolated microspore cultures were induced by different temperatures in two species, by 32ºC in rapeseed, and by 4ºC in barley. Previous studies of our group suggested some factors that would be involved in the microspore embryogenesis initiation and progression. Three of these factors have been analysed in the two species by a multidisciplinary approach: 1) DNA methylation, 2) Arabinogalactan proteins (AGPs) and 3) Endogenous auxin. The results were compared in the two species in order to characterize common mechanisms involved in microspore embryogenesis in monocot and dicot species. We studied their dynamics during the process of microspore embryogenesis, as well as their function by analysing the effect of inhibitors for these factors on the embryogenesis induction and efficiency. In addition, some of them were also analysed during pollen development to identify changes associated with the change of developmental program towards microspore embryogenesis. The results indicated that these factors can be used as early markers of microspore embryogenesis. Since they are found in the two species analysed, independently of the inductor stress, they would inform on general mechanisms involved in the process that could be extended to other plant species. In this thesis, the dynamics of global DNA methylation was studied during microspore embryogenesis and gametophytic development in Hordeum vulgare, and the results were compared with those recently reported by our group in B. napus. The quantifications of global DNA methylation levels and the 5-methyl-deoxycytidine (5mdC) immunofluorescence assays showed low levels of DNA methylation in microspores and a high increase along gametophytic development and pollen maturation. The 5mdC signal increased in the generative and sperm nuclei whereas the vegetative nucleus showed lower signal. After cold stress and microspore embryogenesis induction, low levels of DNA methylation and weak signal of 5mdC were observed in nuclei of responsive microspores and 2 – 4 cell proembryos, indicating that a global hypomethylation occurs during the change of the developmental program and first embryogenic divisions in barley, as it was reported in rapeseed. At later developmental stages, the global DNA methylation was high in developing embryos and it increased during embryo maturation suggesting that DNA methylation is critical for embryo formation and plays a role in the regulation of gene expression in microspore embryogenesis. This thesis also analysed the effects of 5-azacytidine (AzaC), a DNA methylation inhibitor, on microspore embryogenesis initiation and progression in the two species, rapeseed and barley. AzaC treatments decreased global DNA methylation levels and favoured the initiation of microspore embryogenesis, increasing the production of multicellular embryos compared with untreated cultures. In contrast, this agent impaired the progression of microspore embryogenesis, at later stages. Taken together, the results indicate that DNA demethylation by AzaC promotes microspore embryogenesis initiation, while embryo differentiation requires de novo DNA methylation and is prevented by AzaC. The dynamics of the presence and distribution of arabinogalactan proteins (AGPs) were studied during microspore embryogenesis in Brassica napus and Hordeum vulgare, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM 14, JIM 13 and MAC 207). The results showed that AGPs were induced during microspore embryogenesis and the blocking of AGPs with Yariv reagent impaired the process, in both species. This indicates that AGPs play a key role in microspore embryo development. AGPs epitopes were localized on cell walls and cytoplasmic spots suggesting an active production and secretion of AGPs during microspore reprogramming and embryo development. The distribution patterns of AGPs were also analysed during pollen development in Brassica napus and the results indicated that several AGPs are also related to pollen maturation and germination. It was also analysed the expression pattern of the BnAGP Sta – 4 gene in B. napus. The results showed an increase in the expression of the Sta 39 – 4 gene after microspore embryogenesis initiation suggesting that AGPs are induced with the switch of developmental program, constituting early markers of microspore embryogenesis. These results provide new evidence of the role of endogenous AGPs as potential regulating molecules of the process. In this thesis, the dynamics of endogenous auxin was studied during the microspore reprogramming and embryogenesis in the monocot H. vulgare, and the results were compared with those recently reported by our group in B. napus. The results obtained in this thesis provide information about the changes of auxin distribution during microspore embryogenesis in barley, a model system of monocot species. The results showed that auxin increased during early stages of microspore embryogenesis, findings that are consistent with previous results of our group on auxin distribution in microspore embryogenesis of Brassica napus and Quercus suber. It was also analysed, in the present work, the effect of N-1-naphthylphthalamic acid (NPA), inhibitor of polar auxin transport, and P-chlorophenoxyisobutytric acid (PCIB) auxin action inhibitor, on microspore embryogenesis. The analysis revealed that NPA treatment decreased the number of multicellular embryos, after the induction by stress, compared with untreated cultures; PCIB treatments completely inhibited the responsive microspores to reprogram to embryogenesis. These results indicate that polar auxin transport and auxin action have a key role in initiation and progression of microspore embryogenesis in barley, as it was reported in rapeseed, suggesting a common general role of auxin in the process. CONCLUSION In conclusion, the results in this PhD thesis revealed the involvement of DNA methylation, AGPs and auxin in microspore embryogenesis initiation and progression, possibly with common roles in dicot and monocot species. The results suggest that the induction of microspore embryogenesis requires DNA hypomethylation, AGPs induction and auxin accumulation, independently of the inductor stress and the species. Our results could open a way to design new biotechnological strategies for improving doubled-haploid production in breeding programs of horticulture and cereal crops. - Beca del Programa JAE Predoctoral del CSIC (JAEPre 010-052). - Proyectos del Plan Nacional: BFU2008-00203 ; BFU2011-23752 ; AGL2014 – 52028-R -Proyecto de cooperación internacional: Acción integrada Hispano-Portuguesa PRI-AIBPT-2011-0763 El trabajo de esta tesis ha dado lugar a tres publicaciones en revistas JCR (archivos permitidos depositados en Digital.CSIC) y un manuscrito en preparación. 1) Change in DNA methylation levels and nuclear distribution patterns after microspore reprogramming to embryogenesis in barley. http://digital.csic.es/handle/10261/101305 2) 5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley. https://digital.csic.es/handle/10261/118199 3) Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus. https://digital.csic.es/handle/10261/89731. Sin archivo, ver http://dx.doi.org/10.1007/s00497-013-0217-8 4) Auxin increase and distribution, and effects of auxin inhibitors on microspore embryogenesis initiation and progression in barley. (En preparación).

Resumen del trabajo presentad en la XXI Reunión de la Sociedad Española de Fisiología Vegetal y al XIV Congreso Hispano-luso de Fisiología Vegetal, celebradas en Toledo (España) del 14 al 17 de junio de 2015., Microspores are reprogrammed by stress treatments in vitro, changing their developmental pathway to embryogenesis; the process, microspore embryogenesis, represents an important tool in plant breeding to obtain double-haploid plants. DNA methylation is a major epigenetic modification which changes during plant cell differentiation and proliferation, regulating gene expression. Recently, we have shown modifications in global DNA methylation that accompany the microspore reprogramming to embryogenesis. 5-Azacytidine (AzaC), known analog of 5-cytosine, cannot be methylated and leads to DNA hypomethylation in eukaryotic cells, constituting a useful demethylating agent to study DNA epigenetic dynamics, with potential application to improve the efficiency of microspore embryogenesis. This work analyzes for the first time effects of short and long AzaC treatments on microspore embryogenesis induction and progression in two species, the dicot Brassica napus (rapeseed) and the monocot Hordeum vulgare (barley). Quantitative analyses of pro-embryos formed after induction and embryo production, quantification of global DNA methylation levels, 5mdC immunofluorescence at confocal microscope were performed in 2.5 µM AzaC-treated and untreated cultures. Short AzaC treatments produced significant increases of embryogenesis induction rates, by decreasing global DNA methylation levels and modifying 5mdC distribution patterns. In contrast, longer AzaC treatments dramatically diminished embryo production. Similar effects were found in both species indicating that DNA demethylation favors microspore reprogramming, totipotency acquisition and embryogenesis initiation, while subsequent embryo differentiation requires de novo DNA methylation and is impaired by AzaC. Results give new insights into the role of epigenetic mark dynamics by epigenetic inhibitors, like AzaC, in stress-induced microspore embryogenesis, improving its efficiency in biotechnology and agronomic breeding programs., Work supported by projects (BFU2011-23752 and AGL2014-52028-R) funded by the Spanish Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (ERDF/FEDER) of the European Commission.

Trabajo presentado en la XI Reunión de la Sociedad Española de Cultivo in Vitro de Tejidos Vegetales, celebrada en Valencia (España) del 03 al 04 de septiembre de 2015.

The main theme of this paper is the presentation of some critical issues such as transcoding, transport interworking, handover, congestion control and network scalability, that influence the integration of the UMTS and B-ISDN. Solutions to these issues will also be discussed. Furthermore, we will address a number of quantitative (i.e. performance) aspects of an integrated UMTS/B-ISDN system.