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.

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.

Pinus. ponderosa (P. Lawson and C. Lawson) is a commercial tree and one of the most important forest species in North America. Ponderosa pine suffers hardship when going through vegetative propagation and, in some cases, 15–30 years are needed to achieve full reproductive capacity. Based on previous works on P. ponderosa regeneration through in vitro organogenesis and trying to improve the published protocols, our objective was to analyze the influence of different types of explants, basal culture media, cytokinins, auxins, and light treatments on the success of shoot multiplication and rooting phases. Whole zygotic embryos and 44 µΜ 6-benzyladenine showed the best results in terms of explants survival. For shoot organogenesis, whole zygotic embryos and half LP (LP medium, Quoirin and Lepoivre, 1977, modified by Aitken-Christie et al., 1988) macronutrients were selected. A significant positive interaction between whole zygotic embryos and half LP macronutrients was found for the percentage of explants forming shoots. Regarding the light treatments applied, a significantly higher percentage of shoots elongated enough to be rooted was detected in shoots growing under blue LED at a light intensity of 61.09 µmol m−2 s−1. However, the acclimatization percentage was higher in shoots previously cultivated under fluorescent light at a light intensity of 61.71 µmol m−2 s−1. Anatomical studies using light microscopy and scanning electron microscopy showed the light treatments promoted differences in anatomical aspects in in vitro shoots; needles of plantlets exposed to red and blue LEDs revealed less stomata compared with needles from plantlets exposed to fluorescent light.

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 ºC, 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 ºC, 2 h, 10 days; 23 ºC, 10 days) or at a control temperature (23 ºC, 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 ºC 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 ºC. Furthermore, the 5-hmC and 5-hmC/5-mC ratio showed a signi cantly negative correlation with changes in water potential; and a signi cantly 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. Key MessageApplication of high temperatures in the maturation stage of somatic embryos and the resulting Pinus radiata somatic plants provoked a drought adaptation and changes in epigenetic mechanisms.

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.