Ander Castander-Olarieta

Pinus radiata is a commercially valuable forest species whose productivity is increasingly threatened by climate change-related biotic and abiotic stresses, such as diseases, soil salinization, and rising temperatures. Using a sufficiently optimized somatic embryogenesis protocol for radiata pine as a starting point, this study aimed to evaluate the morphological and biochemical responses of embryogenic cell lines subjected to different elicitors, such as methyl jasmonate and tagatose, during proliferation, or to sodium butyrate at the maturation stage. The effect of various stresses, such as salinity and high temperatures, was evaluated at the proliferation and germination stages. Biochemical analyses, including measurements of total protein, hydrogen peroxide, and malondialdehyde contents were carried out in embryogenic tissues and somatic plants, and growth was also evaluated in these tissues and plants. This growth assessment led to the classification of plants according to their suitability for ex vitro planting. Several embryogenic cell lines were tested, and the response to elicitation or stress was found to be highly genotype-dependent; however, some trends were observed, particularly in terms of growth. These results suggest that somatic embryogenesis combined with stress priming or elicitor application may be a viable strategy for improving biotic and abiotic stress response in radiata pine.

Pinus radiata is a commercially valuable forest species whose productivity is increasingly threatened by climate change-related biotic and abiotic stresses such as diseases, soil salinization and rising temperatures. Taking as a starting point a sufficiently optimized somatic embryogenesis propagation protocol for radiata pine, this study aimed to evaluate the morphological and biochemical responses embryogenic cell lines subjected to different elicitors such as methyl jasmonate and tagatose during proliferation or sodium butyrate at maturation stage. The effect of various stresses such as salinity and high temperatures was evaluated at proliferation and germination stages. Then, biochemical analyses (total protein, hydrogene peroxide and malondialdehide contents) were carried out in embryogenic tissues and plants and the growth was also assessed in these tissues and in somatic plants. This latter growth assessment led to a classification of plants depending on their suitability to be planted ex vitro. Several embryogenic cell lines were tested and the response to elicitation or stress was found highly genotype-dependent however some trends could be observed particularly when assessing growth. These results suggest that somatic embryogenesis combined with stress priming or elicitor application may be a viable strategy to enhance biotic and abiotic stress tolerance in radiata pine.

Because of the effects of climate change, a shortage of seeds from superior mother trees for forestry breeding programmes can occur. While biotechnological tools can help address this challenge, the benefits and drawbacks of each technique need to be evaluated. In this work, a comparative Pinus radiata plantlet production study was performed using two different approaches: seeds versus somatic embryos. For each procedure, the number of plantlets obtained was recorded, and the manual labour time was calculated. The skills and time required were higher for somatic embryogenesis, but so was the number of plantlets obtained: 3660 acclimatized somatic plantlets versus 1124 acclimatized zygotic plantlets. The number of different genotypes obtained was higher when germinating mature seeds, but somatic embryogenesis coupled with cryopreservation offers the advantage of having a backup for field-tested material. Additionally, analyses of carbohydrates, total protein and dry weight of somatic and zygotic embryos were carried out. These analyses revealed considerable differences between somatic and zygotic embryos but underlined the importance of high sucrose levels for germination in both systems. In conclusion, each technique offers advantages and disadvantages, and the choice will depend on the objective, the species and the value of the material to be propagated.

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.

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.

Genetic improvement programs for conifer forest species face the challenge of propagating elite individuals with superior characteristics in the present landscape of climate change; the problem is focused on the fact that when these individuals have shown the desirable traits, they have changed phase and therefore have lost the ability to be propagated by traditional methods. Based on our previous works on Pinus spp. regeneration of adult trees through organogenesis and trying to improve the protocol in Pinus radiata, our objective was to analyze the influence of collection dates and different 6-benzyladenine (BA) concentrations in the first phase of shoot induction, as well as the effect of different light types on the success of root induction. Moreover, we were interested in studying the effect of the abovementioned physico-chemical factors on the amino acid and carbohydrate content in the shoots developed in vitro. Reinvigorated shoots were obtained in both BA concentrations (22 or 44 μM), although the highest BA concentration showed the best results in terms of shoot induction (explants forming shoots (46%) and number of shoots per explant (1.95 ± 0.52)) when using initial explants collected in the first week of February. The percentage of explants forming shoots (EFS) was genotype-dependent. Explants from genotype A induced with the highest BA concentration showed the highest EFS (91%). With respect to the light treatment applied, significant differences in root induction (20%) and in the number of roots per explant (4.62 ± 0.65) were observed in shoots cultured under white FL. Finally, significant differences in different phases of the rooting process were detected in the amounts of fructose, glucose and sucrose and in the content of threonine and tyrosine.

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.

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.