Open Access
Induction of radiata pine somatic embryogenesis at high temperatures provokes a long-term decrease in DNA methylation/hydroxymethylation and differential expression of stress-related genes
(MDPI, 2020) Castander Olarieta, Ander; Pereira, Catia; Sales, Ester; Meijón, Mónica; Arrillaga, Isabel; Goicoa Mangado, Tomás; Ugarte Martínez, María Dolores; Cañal, María Jesús; Moncaleán, Paloma; Montalbán, Itziar A.; Estadística, Informática y Matemáticas; Estatistika, Informatika eta Matematika; Institute for Advanced Materials and Mathematics - INAMAT2
Based on the hypothesis that embryo development is a crucial stage for the formation of stable epigenetic marks that could modulate the behaviour of the resulting plants, in this study, radiata pine somatic embryogenesis was induced at high temperatures (23◦ C, eight weeks, control; 40◦ C, 4 h; 60◦ C, 5 min) and the global methylation and hydroxymethylation levels of emerging embryonal masses and somatic plants were analysed using LC-ESI-MS/ MS-MRM. In this context, the expression pattern of six genes previously described as stress-mediators was studied throughout the embryogenic process until plant level to assess whether the observed epigenetic changes could have provoked a sustained alteration of the transcriptome. Results indicated that the highest temperatures led to hypomethylation of both embryonal masses and somatic plants. Moreover, we detected for the first time in a pine species the presence of 5-hydroxymethylcytosine, and revealed its tissue specificity and potential involvement in heat-stress responses. Additionally, a heat shock protein-coding gene showed a down-regulation tendency along the process, with a special emphasis given to embryonal masses at first subculture and ex vitro somatic plants. Likewise, the transcripts of several proteins related with translation, oxidative stress response, and drought resilience were differentially expressed.
Open Access
Temperature and water availability during maturation affect the cytokinins and auxins profile of radiata pine somatic embryos
(Frontiers Media, 2018) Moncaleán, Paloma; García Mendiguren, Olatz; Novák, Ondrej; Strnad,Miroslav; Goicoa Mangado, Tomás; Ugarte Martínez, María Dolores; Montalbán, Itziar A.; Estadística, Informática y Matemáticas; Estatistika, Informatika eta Matematika
Somatic embryogenesis (SE) provides us a potent biotechnological tool to manipulate the physical and chemical conditions (water availability) along the process and to study their effect in the final success in terms of quantity of somatic embryos produced. In the last years, our research team has been focused on the study of different aspects of the SE in Pinus spp. One of the main aspects affecting SE is the composition of culture media; in this sense, phytohormones play one of the most crucial roles in this propagation system. Many studies in conifers have shown that different stages of SE and somatic embryo development are correlated with distinct endogenous phytohormone profiles under the stress conditions needed for the process (i.e., cytokinins play a regulatory role in stress signaling, which it is essential for radiata pine SE). Based on this knowledge, the aim of this study was to test the effect of different temperatures (18, 23, and 28°C) and gelling agent concentrations (8, 9, and 10 gL-1) during the maturation stage of Pinus radiata SE in maturation and germination rates. Parallel, phytohormone profile of somatic embryos developed was evaluated. In this sense, the highest gellan gum concentration led to significantly lower water availability. At this gellan gum concentration and 23°C a significantly higher number of somatic embryos was obtained and the overall success of the process increased with respect to other treatments assayed. The somatic embryos produced in these conditions showed the highest concentration of iP-type cytokinins and total ribosides. Although, the different conditions applied during maturation of somatic embryos led to different hormonal profiles, they did not affect the ex vitro survival of the resulting somatic plants, where no significant differences were observed.
Open Access
Effect of thermal stress on tissue ultrastructure and metabolite profiles during initiation of Radiata pine somatic embryogenesis
(Frontiers Media, 2019) Castander Olarieta, Ander; Montalbán, Itziar A.; Oliveira, Eliana de Medeiros; Dell'Aversana, Emilia; D'Amelia, Luisa; Carillo, Petronia; Steiner, Neusa; Fraga, Hugo Pacheco de Freitas; Guerra, Miguel Pedro; Goicoa Mangado, Tomás; Ugarte Martínez, María Dolores; Estadística, Informática y Matemáticas; Estatistika, Informatika eta Matematika
Climate change will inevitably lead to environmental variations, thus plant drought tolerance will be a determinant factor in the success of plantations and natural forestry recovery. Some metabolites, such as soluble carbohydrates and amino acids, have been described as being the key to both embryogenesis efficiency and abiotic stress response, contributing to phenotypic plasticity and the adaptive capacity of plants. For this reason, our main objectives were to evaluate if the temperature during embryonal mass initiation in radiata pine was critical to the success of somatic embryogenesis, to alter the morphological and ultrastructural organization of embryonal masses at cellular level and to modify the carbohydrate, protein, or amino acid contents. The first SE initiation experiments were carried out at moderate and high temperatures for periods of different durations prior to transfer to the control temperature of 23 degrees C. Cultures initiated at moderate temperatures (30 degrees C, 4 weeks and 40 degrees C, 4 days) showed significantly lower initiation and proliferation rates than those at the control temperature or pulse treatment at high temperatures (50 degrees C, 5 min). No significant differences were observed either for the percentage of embryogenic cell lines that produced somatic embryos, or for the number of somatic embryos per gram of embryonal mass. Based on the results from the first experiments, initiation was carried out at 40 degrees C 4 h; 50 degrees C, 30 min; and a pulse treatment of 60 degrees C, 5 min. No significant differences were found for the initiation or number of established lines or for the maturation of somatic embryos. However, large morphological differences were observed in the mature somatic embryos. At the same time, changes observed at cellular level suggested that strong heat shock treatments may trigger the programmed cell death of embryogenic cells, leading to an early loss of embryogenic potential, and the formation of supernumerary suspensor cells. Finally, among all the differences observed in the metabolic profile, it is worth highlighting the accumulation of tyrosine and isoleucine, both amino acids involved in the synthesis of abiotic stress response-related secondary metabolites.
Open Access
Pinus spp. somatic embryo conversion under high temperature: effect on the morphological and physiological characteristics of plantlets
(MDPI, 2020) Marqués do Nascimento, Antonia Maiara; Barroso, Priscila Alves; Ferreira do Nascimento, Naysa Flavia; Goicoa Mangado, Tomás; Ugarte Martínez, María Dolores; Montalbán, Itziar A.; Moncaleán, Paloma; Estatistika, Informatika eta Matematika; Institute for Advanced Materials and Mathematics - INAMAT2; Estadística, Informática y Matemáticas
Climatic variations in the current environmental scenario require plants with tolerance to sudden changes in temperature and a decrease in water availability. Accordingly, this tolerance will enable successful plantations and the maintenance of natural and planted forests. Consequently, in the last two decades, drought tolerance and high temperatures in conifers have been an important target for morphological, physiological, and epigenetic studies. Based on this, our research team has optimized different stages of somatic embryogenesis (SE) in Pinus spp. improving the success of the process. Through this method, we can obtain a large amount of clonal material and then analyze the somatic plants under different conditions ex vitro. The analysis of the morphological and physiological parameters in somatic embryos (ses) and plants with different tolerances to abiotic stress can provide us with valuable information about the mechanisms used by plants to survive under adverse environmental conditions. Thus, the objective of this work was to evaluate the influence of high temperatures (23, 40, 50, and 60◦C, after 12 weeks, 90, 30, 5 min, respectively) on the morphology of somatic embryos obtained from Pinus radiata D.Don (Radiata pine) and Pinus halepensis Mill. (Aleppo pine). In addition, we carried out a physiological evaluation of the somatic plants of P. radiata submitted to heat and water stress in a greenhouse. We observed that the number of somatic embryos was not affected by maturation temperatures in both species. Likewise, P. radiata plants obtained from these somatic embryos survived drought and heat stress in the greenhouse. In addition, plants originating from embryonal masses (EMs) subjected to high maturation temperature (40 and 60◦C) had a significant increase in gs and E. Therefore, it is possible to modulate the characteristics of somatic plants produced by the manipulation of environmental conditions during the process of SE.

Goicoa Mangado, Tomás

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