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Current climate changes imply an imminent risk for forest species. In this context, somatic embryogenesis is a valuable tool to study the response of plants to different abiotic stresses. Based on this, we applied a high-temperature regime (50 °C, 5 min) during the maturation of Pinus radiata D. Don embryogenic masses in order to evaluate the development of an epigenetic memory months later. Therefore, somatic plants (SP) resulting from somatic embryos (ses) maturated at control temperature and cultivated in a greenhouse were submitted to heat stress (40 °C, 2 h, 10 days; 23 °C, 10 days) or at a control temperature (23 °C, 20 days); while another 20 SP resulting from ses maturated in the two temperature regimes and cultivated in the greenhouse were submitted to drought stress or weekly irrigated. All plants were evaluated for relative water content, water potential, electrolyte leakage, stomatal conductance, transpiration, methylation (5-mC) and hydroxymethylation (5-hmC) levels. The results showed that the SP obtained from ses maturated at 50 °C showed an adaptation to drought stress based on water potential and transpiration. Furthermore, SP kept under heat stress in a greenhouse showed lower 5-hmC levels than SP kept at 23 °C. Furthermore, the 5-hmC and 5-hmC/5-mC ratio showed a significantly negative correlation with changes in water potential; and a significantly negative correlation was observed between the levels of stomatal conductance and 5-mC. We conclude that the manipulation of conditions during the maturation process in somatic embryogenesis modulates the physiological characteristics of the SP obtained. This research was funded by MINECO (Spanish Government) project (AGL2016-76143-C4-3R), CYTED (P117RT0522), and MINECO (BES-2017-081249, 'Ayudas para contratos predoctorales para la formación de doctores'). MULTIFOREVER (Project MULTIFOREVER is supported under the umbrella of ERA-NET cofound Forest Value by ANR (FR), FNR (DE), MINCyT (AR), MINECO-AEI (ES), MMM (FI) and VINNOVA (SE). Forest value has received funding from the European Union's Horizon 2020 research and innovation programmed under Agreement No. 773324.

Somatic embryogenesis (SE) is considered the most effective method for vegetative propagation of Norway spruce (Picea abies L. Karst). For mass propagation, a storage method that is able to handle large quantities of embryogenic tissues (ETs) reliably and at a low cost is required. The aim of the present study was to compare freezing at −80 °C in a freezer to cryopreservation using liquid nitrogen (LN) as a method for storing Norway spruce ETs. The possibility of simplifying both the pre-treatment and thawing processes in cryopreservation was also studied. The addition of abscisic acid (ABA) to the pre-treatment media and using polyethylene glycol PEG4000 instead of PEG6000 in a cryoprotectant solution were tested. Both the pre-and post-treatments on semi-solid media could be simplified by reducing the number of media, without any loss of genotype or embryo production capacity of ETs. On the contrary, the storage of ETs in a freezer at −80 °C instead of using LN was not possible, and the addition of ABA to the pre-treatment media did not provide benefits but increased costs. The lower regeneration rate after using PEG4000 instead of PEG6000 in a cryoprotectant solution in cryovials was unexpected and unwanted. The simplified pre-and post-treatment protocol will remarkably reduce the workload and costs in the mass-cryopreservation of future forest regeneration materials and in thawing the samples for mass propagations, respectively.

La embriogénesis somática es un método prometedor de propagación de coníferas, pero necesita de protocolos optimizados de acuerdo con las diferentes etapas del proceso y la especie modelo. Pinus halepensis Mill. (pino carrasco) es una especie utilizada ampliamente en la reforestación y se logró desarrollar el procedimiento de embriogénesis somática satisfactoriamente pero aun así, existe baja germinación y conversión de embriones somáticos en plantas. En este sentido, promover cambios en el ambiente químico en la etapa de germinación es una alternativa para aumentar las tasas de germinación y la consecuente obtención de plantas somáticas. Teniendo esto en cuenta, el objetivo de este trabajo fue evaluar la influencia de diferentes fuentes de carbohidratos aplicadas durante la etapa de germinación de los embriones somáticos de P. halepensis, sobre el éxito de este proceso y la morfología de las plantas somáticas obtenidas. Se observó un aumento estadísticamente significativo en las tasas de germinación, en la longitud total de las plantas somáticas, así como en la longitud de la raíz principal cuando los embriones somáticos fueron cultivados en el medio de germinación suplementado con maltosa., Somatic embryogenesis is a promising method of propagation of conifers, but it requires optimized protocols according to the different stages of the process and the model species. Pinus halepensis Mill. (Aleppo pine) is a species widely used in reforestation and the somatic embryogenesis procedure was successfully developed, but even so, there is low germination and conversion of somatic embryos into plants. In this sense, promoting changes in the chemical environment in the germination stage is an alternative to increase germination rates and the consequent obtaining of somatic plants. Taking this into account, the objective of this work was to evaluate the influence of different carbohydrate sources applied during the germination stage of the somatic embryos of P. halepensis, on the success of this process and the morphology of the somatic plants obtained. A statistically significant increase in germination rates, total length of somatic plants, as well as principal root length was observed when somatic embryos were cultured in maltosesupplemented germination medium., Facultad de Ciencias Agrarias y Forestales

Somatic embryogenesis (SE) is considered the most effective method for vegetative propagation of Norway spruce (

Among the different in vitro culture techniques, somatic embryogenesis has been one of the most important developments for plant tissue culture; it has enabled mass propagation and the development of biotechnological tools to enhance the productivity and quality of plantation forestry. This propagation technique together with cryopreservation is the base of multivarietal forestry.The development of somatic embryogenesis in forest trees dates from 1985, and in the last years several studies have focused on the development and optimization of the conifer somatic embryogenesis process to make it more efficient in terms of both the quantity and the characteristics of the plants obtained. However, these advances are not sufficiently refined to be implemented commercially for many Pinus spp. due to the high cost of the process derived from hand labor. Nowadays, trying to add value to the plants produced to compensate the high costs of the process, different studies are being developed in order to obtain Pinus somatic plants with better adaptation to environmental stresses prompted by the current situation of climate change.In this chapter, a summary of the recent somatic embryogenesis systems developed to achieve Pinus spp. high quality plants is presented.

Improving the capacity of plants to face adverse environmental conditions requires a deep understanding of the molecular mechanisms governing stress response and adaptation. Proteomics, combined with metabolic analyses, offers a wide resource of information to be used in plant breeding programs. Previous studies have shown that somatic embryogenesis in Pinus spp. is a suitable tool not only to investigate stress response processes but also to modulate the behaviour of somatic plants. Based on this, the objective of this study was to analyse the protein and soluble sugar profiles of Pinus radiata embryonal masses after the application of high temperatures to unravel the mechanisms involved in thermopriming and memory acquisition at early stages of the somatic embryogenesis process. Results confirmed that heat provokes deep readjustments in the life-cycle of proteins, together with a significant reduction in the carbon-flux of central-metabolism pathways. Heat-priming also promotes the accumulation of proteins involved in oxidative stress defence, in the synthesis of specific amino acids such as isoleucine, influences cell division, the organization of the cytoskeleton and cell-walls, and modifies the levels of free soluble sugars like glucose or fructose. All this seems to be regulated by proteins linked with epigenetic, transcriptional and post-transcriptional mechanisms.

Short-term exposure to high temperatures modulates the aromatic cytokinin profile of radiata pine embryonal masses when measured 8 weeks after initiation. Abstract Recent research has demonstrated that cytokinins could be involved in abiotic stress responses. However, little research has been published about the role of endogenous aromatic cytokinins during these processes. Therefore, in this study, different temperature and incubation periods (23 oC, 8 weeks; 40 oC, 4 h; 50 oC, 30 min; 60 oC, 5 min) were applied during induction of radiata pine somatic embryogenesis and the aromatic cytokinin content of the resulting embryonal masses was measured. Results indicated that temperature during induction had a significant effect on the profile of most of the aromatic cytokinins detected. All tested higher temperatures than control provoked a decline in some free bases, including N6-benzyladenine, ortho-Topolin and para-Topolin. On the other hand, the values of N6-benzyladenine nucleotides, ribosides and irreversible metabolites (9G), as well as para-Topolin ribosides, increased in embryonal masses originating from 40 oC treatment. These results suggest that despite not being traditionally considered part of the stress response machinery, aromatic cytokinins might be involved in heat-stress responses during early stages of somatic embryogenesis in radiata pine.

Vegetative propagation through somatic embryogenesis is an effective method to produce elite varieties and can be applied as a tool to study the response of plants to different stresses. Several studies show that environmental changes during embryogenesis could determine future plant development. Moreover, we previously reported that physical and chemical conditions during somatic embryogenesis can determine the protein, hormone and metabolite profiles, as well as the micromorphological and ultrastructural organization of embryonal masses and somatic embryos. In this sense, phytohormones are key players throughout the somatic embryogenesis process as well as during numerous stress-adaptation responses. In this work, we first applied different higherature regimes (30 °C, 4 weeks; 40 °C, 4 days; 50 °C, 5 min) during induction of Pinus radiata D. Don somatic embryogenesis, together with control temperature (23 °C). Then, the somatic plants regenerated from initiated embryogenic cell lines and cultivated in greenhouse conditions were subjected to drought stress and control treatments to evaluate survival, growth and several physiological traits (relative water content, water potential, photosynthesis, stomatal conductance and transpiration). Based on those preliminary results, even more extreme higherature regimes were applied during induction (40 °C, 4 h; 50 °C, 30 min; 60 °C, 5 min) and the corresponding cytokinin profiles of initiated embryonal masses from different lines were analysed. The results showed that the temperature regime during induction had delayed negative effects on drought resilience of somatic plants as indicated by survival, photosynthetic activity and water- use efficiency. However, high temperatures for extended periods of time enhanced subsequent plant growth in well-watered conditions. Higherature regime treatments induced significant differences in the profile of total cytokinin bases, N6-isopentenyladenine, cis-zeatin riboside and trans-zeatin riboside. We concluded that phytohormones could be potential regulators of stress-response processes during initial steps of somatic embryogenesis and that they may have delayed implications in further developmental processes, determining the performance of the generated plants. This research was funded by MINECO (Spanish Government) project (AGL2016-60476143-C4-3R), CYTED (P117RT0522), DECO (Basque government, ‘Ayudas de formación a jóvenes investigadores y tecnólogos’) and MULTIFOREVER (Forest Value, ERANET program, EU). Project MULTIFOREVER is supported under the umbrella of ERA-NET Cofund ForestValue by ANR (FR), FNR (DE), MINCyT (AR), MINECO-AEI (ES), MMM (FI) and VINNOVA (SE). ForestValue has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N°773324. This work was also supported by the Internal Grant Agency of Palacký University (IGA_PrF_2019_020) and from European Regional Development Fund-Project ‘Plants as a tool for sustainable global development’ (No. CZ.02.1.01/0.0/0.0/16_019/0000827).

Changes in the chemical environment at the maturation stage in Pinus spp. somatic embryogenesis will be a determinant factor in the conversion of somatic embryos to plantlets. Furthermore, the study of biochemical and morphological aspects of the somatic embryos could enable the improvement of somatic embryogenesis in Pinus spp. In the present work, the influence of different amino acid combinations, carbohydrate sources, and concentrations at the maturation stage of Pinus radiata D. Don and Pinus halepensis Mill. was analyzed. In P. radiata, the maturation medium supplemented with 175 mM of sucrose and an increase in the amino acid mixture (1,100 mgL–1 of L-glutamine, 1,050 mgL–1 of L-asparagine, 350 mgL–1 of L-arginine, and 35 mgL–1 of L-proline) promoted bigger embryos, with a larger stem diameter and an increase in the number of roots in the germinated somatic embryos, improving the acclimatization success of this species. In P. halepensis, the maturation medium supplemented with 175 mM of maltose improved the germination of somatic embryos. The increase in the amount of amino acids in the maturation medium increased the levels of putrescine in the germinated somatic embryos of P. halepensis. We detected significant differences in the amounts of polyamines between somatic plantlets of P. radiata and P. halepensis; putrescine was less abundant in both species. For the first time, in P. radiata and P. halepensis somatic embryogenesis, we detected the presence of cadaverine, and its concentration changed according to the species.

Organogenesis and somatic embryogenesis have been widely applied as the two main regeneration pathways in plant tissue cultures. However, recalcitrance is still the main restriction in the clonal propagation of many woody species, especially in conifers. They undergo a “phase change” that leads to significant loss of vegetative propagation capacity, reducing the aptitude of tissues and organs to be regenerated in vitro beyond this point. In line with this, the in vitro regeneration of mature conifer trees has been a long-cherished goal in many laboratories worldwide. Based on previous works in Pinus species regeneration from adult trees, we now present data about the culture of apical shoot buds in an attempt to induce organogenesis and somatic embryogenesis to clone mature trees of Aleppo pine (Pinus halepensis). Reinvigorated axillary shoots were submitted to conditions usually applied to induce somatic embryogenesis through the manipulation of culture media, including the use of auxins such as 2,4-Dichlorophenoxyacetic acid and 1-Naphthaleneacetic acid, cytokinins (6-benzyladenine and kinetin), and phytosulfokine (50, 100, and 200 nM). Although somatic embryos could not be obtained, an embryogenic-like tissue was produced, followed by the emergence of actively proliferating non-embryogenic calli. Variations in the consistence, texture, and color of non-embryogenic calli were observed, especially those arising in the media containing phytosulfokine. Reinvigorated shoots, induced by 22 or 44 µM 6-benzyladenine, were obtained through organogenesis and acclimatized, and phenotypically normal plants were obtained.

Organogenesis and somatic embryogenesis have been widely applied as the two main regeneration pathways in plant tissue cultures. However, recalcitrance is still the main restriction in the clonal propagation of many woody species, especially in conifers. They undergo a “phase change” that leads to significant loss of vegetative propagation capacity, reducing the aptitude of tissues and organs to be regenerated in vitro beyond this point. In line with this, the in vitro regeneration of mature conifer trees has been a long-cherished goal in many laboratories worldwide. Based on previous works in Pinus species regeneration from adult trees, we now present data about the culture of apical shoot buds in an attempt to induce organogenesis and somatic embryogenesis to clone mature trees of Aleppo pine (Pinus halepensis). Reinvigorated axillary shoots were submitted to conditions usually applied to induce somatic embryogenesis through the manipulation of culture media, including the use of auxins such as 2,4-Dichlorophenoxyacetic acid and 1-Naphthaleneacetic acid, cytokinins (6-benzyladenine and kinetin), and phytosulfokine (50, 100, and 200 nM). Although somatic embryos could not be obtained, an embryogenic-like tissue was produced, followed by the emergence of actively proliferating non-embryogenic calli. Variations in the consistence, texture, and color of non-embryogenic calli were observed; especially those arising in the media containing phytosulfokine. Reinvigorated shoots, induced by 22 or 44 µM 6-benzyladenine, were obtained through organogenesis and acclimatized, and phenotypically normal plants were obtained.

NEIKER-BRTA INISEFOR Forest 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, España Instituto de Investigación y Servicios Forestales

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 °C for 4 h, 50 °C for 30 min, and 60 °C 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.

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 &times, 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 &minus, 80 &deg, C and regenerated after one and five years.

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 &deg, 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 &deg, 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.

Owing to the increasing human population and the increasing global demand for wood, its consumption is exceeding the natural rate of regeneration in many areas worldwide. Despite only 3% of the world’s forested land is plantation forest, plantations are highly productive; and with further improvement in genetic composition of planting stock as well as applying biotechnology, additional productivity increases can be obtained. For this reason, it is necessary to enrich traditional breeding programs with biotechnological tools able to increase the quantity and quality of the forestry plants produced. FAO’s definition of forest biotechnology encompasses different techniques for cloning forest trees. Forestry companies are currently considering clonal propagation as a good source of forestry plants. Clonal propagation can be achieved by various means: grafting, rooting of cuttings, coppicing, or in vitro propagation. Several methods of clonal propagation are being practiced with conifers. Along this chapter, a summary of some of the different approaches to improve Pinus spp. clonal propagation will be described, particularly those made in our laboratory.

Climate change is leading to higher temperatures and lower precipitation; this fact can have a negative impact on plant performance and survival. Latest findings have revealed that the conditions in which zygotic embryogenesis takes place have an impact on the adaptive capacity of the resulting plants. Somatic embryogenesis 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 of the process in terms of quantity and quality of somatic plants produced. The development of somatic cells to somatic plantlets comprises three stages: induction of embryonal masses, maturation of embryogenic tissues, and conversion into somatic plants. Our experience in somatic embryogenesis in Pinus spp. enables us to explore the possibility to modulate the quality in terms of abiotic stress tolerance of somatic plants modifying environmental conditions during the initial stages of the process. Our results have shown that the modification of environmental conditions affected not only the success of the process in some species of pines but also the water use efficiency of the somatic plants after several months in ex vitro conditions. In the chapter, we will show the different responses obtained in all the stages of the somatic embryogenesis process as well as the response obtained after drought periods in plants growing in the greenhouse under ex vitro conditions.

This is the first report of (1) somatic embryogenesis in the hybrids of Pinus attenuata with Pinus radiata var. cedrosensis and (2) use of Pinus radiata somatic embryogenic tissue as nurse tissue to facilitate initiation from immature hybrid pine zygotic embryos. Embryogenic cell lines were successfully initiated from first generation reciprocal hybrids of Pinus radiata var. cedrosensis and Pinus attenuata, using P. radiata protocols. This is also the first report of P. radiata somatic embryogenic tissue used as a nurse to facilitate initiation from excised zygotic embryos. Two media formulations were tested, Glitz and Glitz2. Both media are based on a modified Litvay medium. The medium Glitz differs from Glitz2 in absence of casein, an increase in l-glutamine concentration and addition of asparagine. Overall analyses of treatment effects showed the excised zygotic embryo treatments, with or without the nurse on Glitz medium, were significantly better for cell line establishment (47 and 45 %, respectively) than either of the megametophyte treatments or the Glitz2 excised embryo treatment (p ≤ 0.05). Total cell line establishment irrespective of hybrid cross combination, collection time and cultural treatment was 32 %. Establishment percentages within each of the reciprocal hybrids, five crosses of each, across all four collections and cultural treatments, were not significantly different despite significant differences in zygotic embryo development (p ≤ 0.05). From response to the initiation treatments tested in this research, P. attenuata appears to be a much better mother than P. radiata var. cedrosensis with 39 % of cell lines established compared to only 23 % from P. radiata var. cedrosensis. However, analyses of variance results did not indicate that this difference between the two hybrid types was significant, probably due to the large amount of variation among the individual crosses within each of the hybrid types.

Pinus halepensis has been described as a drought-tolerant species with high plasticity to growth in different environments. Its eco-physiological characteristics could facilitate the use of this species in large afforestations in the future scenery of climate change. Somatic embryogenesis is a biotechnological tool with potential for large-scale clonal propagation. In order to establish an improved regeneration protocol for Pinus halepensis, the effects of different temperatures (18, 23, and 28 °C) and water availability conditions (2, 3, and 4 g L−1 Gelrite®), during initiation of embryonal masses on the rate of initiation, proliferation, maturation, and the number of embryos developed, were evaluated. It was found that environmental conditions during the initiation stage of Pinus halepensis somatic embryogenesis influence the success of initiation and proliferation. In contrast, there was no effect of these conditions on the maturation rates and the number of somatic embryos. Somatic embryos were obtained in all treatments tested, indicating that plants can be produced from extreme conditions of induction, such as high temperatures (28 °C) and low water availability conditions (4 g L−1).

Physicochemical conditions present during the initiation of somatic embryogenesis in Pinus radiata determine the rate of embryogenic cell lines generated as well as the final number of somatic embryos. The effect of physical and chemical conditions during the initiation and proliferation stages of somatic embryogenesis in radiata pine was investigated. The first objective was to assess if different temperatures and/or water availability during the initiation or proliferation of embryogenic tissue impacted the success rate during each stage of somatic embryogenesis. The second objective was to determine what stage has a greater influence on subsequent stages of development. Some treatments (18 °C, 4 g L−1 gellan gum) resulted in a higher percentage of initiation whereas others (28 °C, 2 g L−1 gellan gum) gave rise to lower initiation. Our results indicated that initiation at different temperatures affected the subsequent stages of somatic embryogenesis. Thus, these embryogenic tissues were influenced by the environmental conditions present at initiation. We were able to successfully regenerate somatic embryos from cell lines initiated under all the environmental conditions tested.

Aleppo pine (Pinus halepensis Mill.), a native species of the Mediterranean region, has been suggested as a species that when introduced in degraded areas could facilitate the long-term colonization and expansion of late-successional species. Due to climate changes, plants need to withstand extreme environmental conditions through adaptation and changings in developmental pathways. Among other paths, plants undergo changes in developmental pathways controlled by phytohormones. At the same time, somatic embryogenesis has been widely used as a model to understand the mechanisms involved in plant response to different stresses. In this study, in order to induce a strong effect of temperature stress on plants regenerated from somatic embryos, higher temperatures (40 &deg, C for 4 h, 50 &deg, C for 30 min, and 60 &deg, C for 5 min) than the control (23 &deg, C) were applied during the induction stage of somatic embryogenesis in Pinus halepensis. A morphological characterization of the embryogenic cultures showed small differences in the number of starch grains, lipid bodies, and phenolic compounds between treatments. Results showed that high temperatures (60 &deg, C) led to higher rates at the maturation stage of somatic embryogenesis when compared to the control (23 &deg, C), strengthening the productivity through the increase in the number of somatic embryos obtained. Finally, analysis of endogenous concentration of cytokinins showed that different conditions applied during the initiation phase of somatic embryogenesis led to different hormonal profiles, isoprenoid cytokinins showed a clear defined pattern with the higher total hormone concentration being found in embryonal masses induced at 50 &deg, C for 30 min, while different aromatic cytokinins presented different individual responses to the treatments applied. These differences corroborate the idea that cytokinins could be potential regulators of stress&ndash, response processes during initial steps of somatic embryogenesis.

Radiata pine ( Pinus radiata D. Don) is one of the most widely planted exotic pine species in rainfall environments of the Southern hemisphere (Yan et al. 2006). Its fast growth has stimulated an exhaustive study of wood production, and the development of breeding programs (Espinel et al. 1995; Codesido and Fernandez-Lopez 2009). Although utility of in vitro organogenesis has been proven for clonal propagation of this species (Aitken-Christie et al. 1985), a limitation of this method is the high cost of the process for mass production commercially.

Aleppo pine (Pinus halepensis Mill.) is a medium size tree (20–40 m) that can display a shrubby phenotype when growing on harsh conditions (Eckenwalder, 2009). Female cones

Aim of the study: The effect of physical and chemical conditions at proliferation stage was evaluated in order to elucidate if this stage is the determinant phase to induce a marked effect in Pinus halepensis somatic embryogenesis. Area of study : The study was conducted in research laboratories of Neiker (Arkaute, Spain). Material and methods: Pinus halepensis embryonal masses from ten embryogenic cell lines subjected to nine treatments (tissues cultured at three temperatures on media supplemented with three agar concentrations) at proliferation stage. Main results: Significant differences were observed among different proliferation conditions months later at the end of maturation, germination and acclimatization stages. Research highlights: Aleppo pine embryonal masses are cultured under standard conditions on a culture medium supplemented with 4.5 g/L Gelrite® at 23oC. However, better results in terms of plantlet production can be obtained proliferating the embryonal masses at 18oC in a culture media with significantly lower water availability.

Traditionally, many efforts have been carried out in order to improve the success of somatic embryogenesis process in conifers, but little attention has been paid to the influence of the plant material storage conditions in the subsequent phases of the somatic embryogenesis process. In this work our objective was to study the feasibility of storing Pinus radiata plant material at 4 °C for long periods in order to make easier the initiation with high amount of cell lines. The effect of cold storage on the different stages of somatic embryogenesis process has been evaluated. Storage periods of 1–3 months enhanced initiation rates and the number of somatic embryos obtained in the embryogenic cell lines. These results demonstrate the beneficial effect of cold storage and open the possibility of considering a cold preconditioning of plant material as a good alternative to improve the somatic embryogenesis process in conifers.

Pinus halepensis Mill. is a common forest species in the Mediterranean area and it is important for environmental conservation. This study established a method of regenerating Pinus halepensis Mill. through somatic embryogenesis. The effect of culture medium (mineral salts, nitrogen source and plant growth regulators), collection date and seed family on embryogenic tissue initiation and proliferation in Pinus halepensis was analysed during the first steps of embryogenesis process. This study showed a marked effect of the culture medium tested as well as some significant differences among collection dates. Furthermore, the embryogenic tissue initiation was affected by the amino acid mixture in the culture medium and the proliferation stage was significantly affected by the combination of plant growth regulators. At the end of the maturation phase the presence of activated charcoal was also evaluated. Finally, maturation of embryogenic tissue was affected by the nitrogen source in the culture medium and these results were different for high and low mature embryo producing cell lines. To the best of our knowledge, this is the first report on Aleppo pine somatic embryogenesis describing a simple and efficient procedure for large-scale somatic embryo production.

In Pinus radiata D. Don, the transition from the juvenile to the mature phase is characterized by a reduction in the tree's organogenic potential, which is usually reverted in breeding programs by reinvigoration procedures to enable vegetative propagation. In this work, we have determined the best culture conditions for in vitro reinvigoration of radiata pine buds, tested different cytokinin (CK) types [N⁶-benzyladenine (BA), meta-topolin (mT) and trans-zeatin] and concentrations (25 and 50 µM), and studied the effect of culture conditions on endogenous CK and indole-3-acetic acid (IAA) levels at different stages of the organogenic process. To this end, the levels of 43 CKs and IAA were determined in P. radiata buds before and during the reinvigoration process. When BA or mT was applied to the induction medium, we did not observe any significant increase or decrease in endogenous isoprenoid CK content. We also report for the first time the presence of O-glucosides in non-treated P. radiata explants from the field and remark the importance of O-glucosides as storage forms.

Somatic embryogenesis (SE) has been the most important development for plant tissue culture, not only for mass propagation but also for enabling the implementation of biotechnological tools that can be used to increase the productivity and wood quality of plantation forestry. Development of SE in forest trees started in 1985 and nowadays many studies are focused on the optimization of conifer SE system. However, these advances for many Pinus spp. are not sufficiently refined to be implemented commercially. In this chapter, a summary of the main systems used to achieve SE in Pinus spp. is reported.

Somatic embryogenesis (SE) has been the most important development for plant tissue culture, not only for mass propagation but also for enabling the implementation of biotechnological tools that can be used to increase the productivity and wood quality of plantation forestry. Development of SE in forest trees started in 1985 and nowadays many studies are focused on the optimization of conifer SE system. However, these advances for many Pinus spp. are not sufficiently refined to be implemented commercially. In this chapter, a summary of the main systems used to achieve SE in Pinus spp. is reported.