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

The chemical environment at germination stage in Pinus halepensis somatic embryogenesis: implications in the morphological characteristics of the developed plantlets

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 Forestale

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

Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Profile of Radiata Pine Somatic Embryos

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

The effect of changing temperature and agar concentration at proliferation stage in the final success of Aleppo pine somatic embryogenesis

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.MINECO, Spanish Government (project AGL2013-4700-C4-2R)