Pinus canariensis plant regeneration through somatic embryogenesis

Aim of the study: To develop an efficient method to regenerate plants through somatic embryogenesis of an ecologically relevant tree species such as Pinus canariensis.Area of study: The study was conducted in the research laboratories of Neiker-Tecnalia (Arkaute, Spain).Material and methods: Green cones of Pinus canariensis from two collection dates were processed and the resulting immature zygotic embryos were cultured on three basal media. The initiated embryogenic tissues were proliferated testing two subculture frequencies, and the obtained embryogenic cell lines were subjected to maturation. Germination of the produced somatic embryos was conducted and acclimatization was carried out in a greenhouse under controlled conditions.Main results: Actively proliferating embryogenic cell lines were obtained and well-formed somatic embryos that successfully germinated were acclimatized in the greenhouse showing a proper growth.Research highlights: This is the first report on Pinus canariensis somatic embryogenesis, opening the way for a powerful biotechnological tool for both research purposes and massive vegetative propagation of this species.Keywords: acclimatization; Canary Island pine; micropropagation; embryogenic tissue; somatic embryo.Abbreviations used: embryogenic tissue (ET); established cell line (ECL);  somatic embryogenesis (SE); somatic embryos (Se’s)

Heat Stress in Pinus halepensis Somatic Embryogenesis Induction: Effect in DNA Methylation and Differential Expression of Stress-Related Genes

In the current context of climate change, plants need to develop different mechanisms of stress tolerance and adaptation to cope with changing environmental conditions. Temperature is one of the most important abiotic stresses that forest trees have to overcome. Recent research developed in our laboratory demonstrated that high temperatures during different stages of conifer somatic embryogenesis (SE) modify subsequent phases of the process and the behavior of the resulting ex vitro somatic plants. For this reason, Aleppo pine SE was induced under different heat stress treatments (40 \u00b0C for 4 h, 50 \u00b0C for 30 min, and 60 \u00b0C for 5 min) in order to analyze its effect on the global DNA methylation rates and the differential expression of four stress-related genes at different stages of the SE process. Results showed that a slight decrease of DNA methylation at proliferating embryonal masses (EMs) can correlate with the final efficiency of the process. Additionally, different expression patterns for stress-related genes were found in EMs and needles from the in vitro somatic plants obtained; the DEHYDRATION INDUCED PROTEIN 19 gene was up-regulated in response to heat at proliferating EMs, whereas HSP20 FAMILY PROTEIN and SUPEROXIDE DISMUTASE [Cu-Zn] were down-regulated in needles

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

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

Hybrid pine (Pinus attenuata × Pinus radiata) somatic embryogenesis: what do you prefer, mother or nurse?

Development of hybrid pines of Pinus radiata D. Don for commercial forestry presents an opportunity to diversify the current resource of plant material. Climate change and different land uses pose challenges, making alternative species necessary to guarantee wood and non-wood products in the future. Pinus radiata var. cedrosensis × Pinus attenuata hybrid possesses different attributes, such as tolerance to drought conditions, better growth and resistance to snow damage at higher altitudes, and more importantly, different wood quality characteristics. Embryogenic cell lines were successfully initiated reciprocal hybrids using as initial explants megagametophytes, excised zygotic embryos and excised zygotic embryos plus nurse culture. However, the questions raised were: does the initiation environment affect the conversion to somatic plantlets months later? Does the mother tree or the cross have an effect on the conversion to somatic plantlets? In the present work we analysed the maturation rate, number of somatic embryos, germination rate, and the ex-vitro growth in cell lines derived from different initiation treatments, mother tree species, and crosses. Differences were not observed for in vitro parameters such as maturation and germination. However, significant differences were observed due to the mother tree species in relation with the ex-vitro growth rates observed, being higher those in which P. radiata acted as a mother. Moreover, embryogenic cell lines from these hybrids were stored at −80◦C and regenerated after one and five years.This research was funded by MINECO (Spanish Government) project (AGL2016-76143-C4-3R), CYTED (P117RT0522), DECO (Basque government, 'Ayudas de formación a jóvenes investi-gadores y tecnólogos'). OECD Co-operative Research Programme Fellowship (Biological Resource Management for Sustainable Agricultural Systems, 2013) for supporting the visit of Paloma Moncaleán to Scion and Scion Core Funding for supporting the hybrid pine initiative

Effect of Thermal Stress on Tissue Ultrastructure and Metabolite Profiles During Initiation of Radiata Pine Somatic Embryogenesis

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°C. Cultures initiated at moderate temperatures (30°C, 4 weeks and 40°C, 4 days) showed significantly lower initiation and proliferation rates than those at the control temperature or pulse treatment at high temperatures (50°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°C 4 h; 50°C, 30 min; and a pulse treatment of 60°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

Optimization of the micropropagation of elite adult trees of Sequoia sempervirens: forest species of interest in the Basque Country, Spain

NEIKER-BRTA INISEFORForest trees are renewable sources of timber and other valuable non-timber products. Nowadays, the increase in population and demand for forestry products results in overexploitation of forestry. Therefore, there is an urgent need to produce elite plants with higher productivity under stress derived from climate change to have available to afforestation. For this reason, propagation methods should be improved to be more efficient in terms of quality and productivity. The main species planted in the Basque Country is Pinus radiata; during the last three years, Pinus radiata plantations have suffered a fungus attack affecting mainly needles until the tree's death. This crisis is caused by the combined action of two fungi of the genus Dothistroma and Lecanosticta acicola, whose expansion seems to have been enhanced by weather-related factors, such as humid and hot summers. Although we have evidence of this disease's presence in our mountains since 1942, the disease has had a speedy expansion with an aggressive effect for reasons that are not scientifically known today. For the above, Basque Country forestry sector is looking for alternative species to be used in its plantations. Part of the forestry sector considers that Sequoia sempervirens could be a good choice for plantations. Besides, its high-quality wood and its tolerance to the attack of several pathogens and other diseases derived from climate change are characteristics that could confer some advantage over other forest species. The main goal of this study was to optimize the micropropagation of adult elite trees of Sequoia sempervirens. The effect of 6-benzylaminopurine, meta-Topolin and Kinetin, and 4 types of explant in the multiplication stage were analyzed to carry out this objective. Furthermore, the effect of two types of auxins: 1-naphthalene acetic acid, indole-3-butyric acid, and a mixture of both, were evaluated on the induction of roots and their subsequent effect on the acclimatization process. The best multiplication index was obtained when 4.4 µM 6-benzylaminopurine and apical explants longer than 1.5 cm of length were used. The root induction percentage was 75% in the most responsive genotype analyzed when 4.4 µM 6-benzylaminopurine was used on the induction stage, and 50 µM of 1-naphthalene acetic acid was used for rooting. Finally, after 3 months in the greenhouse, the explants cultured with Kinetin and rooted in a culture medium with indole-3-butyric acid showed the highest acclimatization success (94%).Los árboles forestales son fuentes renovables de madera y otros valiosos productos no maderables. Hoy en día, el aumento de la población y la demanda de productos forestales se traduce en una sobreexplotación forestal. Por lo tanto, existe una necesidad urgente de producir plantas de élite con mayor productividad bajo estrés derivado del cambio climático para tener disponibles para la forestación. Por esta razón, los métodos de propagación deben mejorarse para que sean más eficientes en términos de calidad y productividad. La principal especie plantada en el País Vasco es Pinus radiata; durante los últimos tres años, las plantaciones de Pinus radiata han sufrido un ataque de hongos que afectó principalmente a las agujas hasta la muerte del árbol. Esta crisis está provocada por la acción combinada de dos hongos del género Dothistroma y Lecanosticta acicola, cuya expansión parece haberse visto favorecida por factores meteorológicos, como los veranos húmedos y calurosos. Si bien tenemos evidencia de la presencia de esta enfermedad en nuestras montañas desde 1942, la enfermedad ha tenido una rápida expansión con un efecto agresivo por razones que no se conocen científicamente en la actualidad. Por lo anterior, el sector forestal del País Vasco está buscando especies alternativas para ser utilizadas en sus plantaciones. Parte del sector forestal considera que Sequoia sempervirens podría ser una buena opción para plantaciones. Además, su madera de alta calidad y su tolerancia al ataque de varios patógenos y otras enfermedades derivadas del cambio climático son características que podrían conferir alguna ventaja sobre otras especies forestales. El objetivo principal de este estudio fue optimizar la micropropagación de árboles adultos de élite de Sequoia sempervirens. Para llevar a cabo este objetivo se analizó el efecto de la 6-bencilaminopurina, meta-Topolin y Kinetin, y 4 tipos de explantes en la etapa de multiplicación. Además, se evaluó el efecto de dos tipos de auxinas: ácido 1-naftaleno acético, ácido indol-3-butírico y una mezcla de ambos sobre la inducción de raíces y su efecto posterior en el proceso de aclimatación. El mejor índice de multiplicación se obtuvo cuando se utilizaron 4,4 µM de 6-bencilaminopurina y explantes apicales de más de 1,5 cm de longitud. El porcentaje de inducción de la raíz fue del 75% en el genotipo más sensible analizado cuando se usó 4,4 µM de 6-bencilaminopurina en la etapa de inducción y 50 µM de ácido 1-naftaleno acético para el enraizamiento. Finalmente, luego de 3 meses en invernadero, los explantes cultivados con Kinetin y enraizados en un medio de cultivo con ácido indol-3-butírico mostraron el mayor éxito de aclimatación (94%)As árvores florestais são fontes renováveis ​​de madeira e outros produtos não-madeireiros valiosos. Hoje, o aumento da população e da demanda por produtos florestais está levando à superexploração das florestas. Portanto, há uma necessidade urgente de produzir plantas de elite com maior produtividade sob estresse das mudanças climáticas para disponibilizá-las para florestamento. Por isso, os métodos de propagação devem ser aprimorados para serem mais eficientes em termos de qualidade e produtividade. As principais espécies plantadas no País Basco são Pinus radiata; Nos últimos três anos, as plantações de Pinus radiata sofreram um ataque de fungos que afetou principalmente as agulhas até a morte da árvore. Essa crise é causada pela ação conjunta de dois fungos do gênero Dothistroma e Lecanosticta acicola, cuja expansão parece ter sido favorecida por fatores meteorológicos, como verões quentes e úmidos. Embora tenhamos evidências da presença desta doença em nossas montanhas desde 1942, a doença tem se expandido rapidamente com efeitos agressivos por razões que não são cientificamente compreendidas no momento. Portanto, o setor florestal do País Basco está em busca de espécies alternativas para serem utilizadas em seus plantios. Parte do setor florestal considera que a Sequoia sempervirens pode ser uma boa opção de plantio. Além disso, sua madeira de alta qualidade e sua tolerância ao ataque de vários patógenos e outras doenças derivadas das mudanças climáticas são características que podem conferir uma vantagem sobre outras espécies florestais. O objetivo principal deste estudo foi otimizar a micropropagação de árvores adultas de Sequoia sempervirens elite. Para a realização deste objetivo, foram analisados ​​os efeitos da 6-benzilaminopurina, meta-Topolina e Kinetina, além de 4 tipos de explantes em fase de multiplicação. Além disso, foi avaliado o efeito de dois tipos de auxinas: ácido 1-naftaleno acético, ácido indol-3-butírico e uma mistura de ambos na indução de raízes e seu posterior efeito no processo de aclimatação. O melhor índice de multiplicação foi obtido quando 4,4 µM de 6-benzilaminopurina e explantes apicais com mais de 1,5 cm de comprimento foram usados. A porcentagem de indução radicular foi de 75% no genótipo mais sensível analisado quando 4,4 µM de 6-benzilaminopurina foi usado na fase de indução e 50 µM de ácido 1-naftaleno acético para enraizamento. Finalmente, após 3 meses em casa de vegetação, os explantes cultivados com Kinetin e enraizados em meio de cultura com ácido indol-3-butírico apresentaram o maior sucesso de aclimatação (94%).Universidad Nacional, Costa Rica.NEIKER, Instituto Vasco de Investigación y Desarrollo Agrario, EspañaInstituto de Investigación y Servicios Forestale

Proteomic and Metabolic Analysis of <i>Pinus halepensis</i> Mill. Embryonal Masses Induced under Heat Stress

Understanding the physiological and molecular adjustments occurring during tree stress response is of great importance for forest management and breeding programs. Somatic embryogenesis has been used as a model system to analyze various processes occurring during embryo development, including stress response mechanisms. In addition, “priming” plants with heat stress during somatic embryogenesis seems to favor the acquisition of plant resilience to extreme temperature conditions. In this sense, Pinus halepensis somatic embryogenesis was induced under different heat stress treatments (40 °C for 4 h, 50 °C for 30 min, and 60 °C for 5 min) and its effects on the proteome and the relative concentration of soluble sugars, sugar alcohols and amino acids of the embryonal masses obtained were assessed. Heat severely affected the production of proteins, and 27 proteins related to heat stress response were identified; the majority of the proteins with increased amounts in embryonal masses induced at higher temperatures consisted of enzymes involved in the regulation of metabolism (glycolysis, the tricarboxylic acid cycle, amino acid biosynthesis and flavonoids formation), DNA binding, cell division, transcription regulation and the life-cycle of proteins. Finally, significant differences in the concentrations of sucrose and amino acids, such as glutamine, glycine and cysteine, were found