Dataset Information

New Approaches to Optimize Somatic Embryogenesis in Maritime Pine.

ABSTRACT: Maritime pine (Pinus pinaster Aiton) is a coniferous native of the Mediterranean basin. Because of its adaptability to a wide range of environmental conditions, the species have become a model for studies in coniferous forest management and functional genomics. Somatic embryogenesis (SE) has been so far, the preferred biotechnological strategy for maritime pine breeding programs initiated at the middle-end of the 20th century. To overcome the limitations of the induction and maturation phases in maritime pine SE, we analyzed the possible maternal influence on the embryogenic capability of megagametophytes from controlled crosses, as well as the effect of the temperature and water availability during SE process on the production of plants. A strong maternal effect on the embryogenic potential of maritime pine megagametophytes was observed in our experiments using half-sib and full-sib progenies, while paternal effect was almost undetectable. Besides, it seems possible to improve somatic embryo production of maritime pine megagametophytes by adjusting optimal temperature throughout the process: 28°C during induction and proliferation, and 23°C during the maturation phase. Using induction and proliferation media with reduced water availability (6 g/L Gelrite) can also increase embryo production. Since other limitation of maritime pine SE is culture decline of embryogenic masses (EMs), that reduces embryo yield and germination, we assessed the profile of ABA and IAA and the expression of two embryogenesis-related genes (LEC1 and WOX2) during maturation of EMs of two morphotypes that differed in their maturation capability. Spiky morphotype (SK), with high maturation capability, had a steady increase in both hormones along the 12 weeks of the maturation, whereas ABA content in smooth morphotype picked at the 4th week and dropped. EMs with this morphotype also had a higher IAA content at the beginning of the maturation. A decrease of LEC1 and WOX2 gene expression over the course of embryo development was found to be characteristic of the SK with high maturation capability.

SUBMITTER: Arrillaga I

PROVIDER: S-EPMC6389691 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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New Approaches to Optimize Somatic Embryogenesis in Maritime Pine.

Arrillaga Isabel I Morcillo Marian M Zanón Israel I Lario Francisco F Segura Juan J Sales Ester E

Frontiers in plant science 20190219

Maritime pine (<i>Pinus pinaster</i> Aiton) is a coniferous native of the Mediterranean basin. Because of its adaptability to a wide range of environmental conditions, the species have become a model for studies in coniferous forest management and functional genomics. Somatic embryogenesis (SE) has been so far, the preferred biotechnological strategy for maritime pine breeding programs initiated at the middle-end of the 20th century. To overcome the limitations of the induction and maturation ph ...[more]

PMID: 30838010

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Project description:The significant morphological differences observed during embryo development in angiosperms and gymnosperms are expected to be the result of a differential control of gene expression. We used a loblolly pine (Pinus taeda) cDNA microarray to analyze global transcriptional changes along zygotic embryogenesis in maritime pine (Pinus pinaster). A time-course analysis of the data obtained from the five embryo developmental groups used in this study led to the identification of 4,645 genes whose expression varied along P. pinaster embryogenesis. These transcripts were clustered into six distinct expression profiles. The grouping of these profiles in early, mid-embryogenesis and embryo maturation, according to the developmental period where most of the sequences were up-regulated, evidenced that characteristic transcriptional changes are associated to each developmental period. The application of a cut-off value of 1.95-fold change led to the identification of 1,838 differentially expressed transcripts that were categorized by biological process. Metabolism, interaction with the environment, oxidation-reduction and transport were some of the most represented categories. During early embryogenesis genes putatively involved in phytohormone-mediated signaling were identified, whereas in middle stages the overrepresented genes could be associated with cotyledon formation and induction of somatic embryogenesis. Genes associated with the synthesis of storage products were up-regulated in the latest stages of pine embryo development. It was also during this developmental period that the largest number of sequences putatively encoding transcription factors was identified. Pine immature zygotic embryos were pooled into five different developmental groups according to the collection date (Day0; Day5; Day11; Day15 and Day25). A common reference design was used. Total RNA was extracted from each sample. Three extractions were prepared per sample (biological replicates). A defined ammount from each RNA sample was pooled to use as reference. Messenger RNA was amplified for both test samples and reference. These were hybridized together in two separate experiments (technical replicates). In total, 30 slides were analyzed.

Project description:BackgroundPredicting the adaptability of forest tree populations under future climates requires a better knowledge of both the adaptive significance and evolvability of measurable key traits. Phenotypic plasticity, standing genetic variation and degree of phenotypic integration shape the actual and future population genetic structure, but empirical estimations in forest tree species are still extremely scarce. We analysed 11 maritime pine populations covering the distribution range of the species (119 families and 8 trees/family, ca. 1300 trees) in a common garden experiment planted at two sites with contrasting productivity. We used plant height as a surrogate of fitness and measured five traits (mean and plasticity of carbon isotope discrimination, specific leaf area, needle biomass, Phenology growth index) related to four different strategies (acquisitive economics, photosynthetic organ size, growth allocation and avoidance of water stress).ResultsEstimated values of additive genetic variation would allow adaptation of the populations to future environmental conditions. Overall phenotypic integration and selection gradients were higher at the high productivity site, while phenotypic integration within populations was higher at the low productivity site. Response to selection was related mainly to photosynthetic organ size and drought-avoidance mechanisms rather than to water use efficiency. Phenotypic plasticity of water use efficiency could be maladaptive, resulting from selection for height growth.ConclusionsContrary to the expectations in a drought tolerant species, our study suggests that variation in traits related to photosynthetic organ size and acquisitive investment of resources drive phenotypic selection across and within maritime pine populations. Both genetic variation and evolvability of key adaptive traits were considerably high, including plasticity of water use efficiency. These characteristics would enable a relatively fast micro-evolution of populations in response to the ongoing climate changes. Moreover, differentiation among populations in the studied traits would increase under the expected more productive future Atlantic conditions.

Project description: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.

Project description:BACKGROUND: Pinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases. DESCRIPTION: EuroPineDB is the largest sequence collection available for a single pine species, Pinus pinaster (maritime pine), since it comprises 951 641 raw sequence reads obtained from non-normalised cDNA libraries and high-throughput sequencing from adult (xylem, phloem, roots, stem, needles, cones, strobili) and embryonic (germinated embryos, buds, callus) maritime pine tissues. Using open-source tools, sequences were optimally pre-processed, assembled, and extensively annotated (GO, EC and KEGG terms, descriptions, SNPs, SSRs, ORFs and InterPro codes). As a result, a 10.5× P. pinaster genome was covered and assembled in 55 322 UniGenes. A total of 32 919 (59.5%) of P. pinaster UniGenes were annotated with at least one description, revealing at least 18 466 different genes. The complete database, which is designed to be scalable, maintainable, and expandable, is freely available at: http://www.scbi.uma.es/pindb/. It can be retrieved by gene libraries, pine species, annotations, UniGenes and microarrays (i.e., the sequences are distributed in two-colour microarrays; this is the only conifer database that provides this information) and will be periodically updated. Small assemblies can be viewed using a dedicated visualisation tool that connects them with SNPs. Any sequence or annotation set shown on-screen can be downloaded. Retrieval mechanisms for sequences and gene annotations are provided. CONCLUSIONS: The EuroPineDB with its integrated information can be used to reveal new knowledge, offers an easy-to-use collection of information to directly support experimental work (including microarray hybridisation), and provides deeper knowledge on the maritime pine transcriptome.

Dataset Information

New Approaches to Optimize Somatic Embryogenesis in Maritime Pine.

Publications

New Approaches to Optimize Somatic Embryogenesis in Maritime Pine.

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