[Show abstract][Hide abstract] ABSTRACT: Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.
[Show abstract][Hide abstract] ABSTRACT: Maritime pine somatic embryos require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome (2D-SDS-PAGE with MS identification) of immature somatic embryos, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous ABA (GC-MS), soluble sugars (HPLC), starch, and confocal laser microscope observations. This multi-scale, integrated analysis was used to unravel early molecular and physiological events involved in somatic embryo development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation which may be antagonistic to somatic embryo development. Under favorable conditions (9G), somatic embryos adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of somatic embryo development whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in development of pine somatic embryos following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.
Physiologia Plantarum 01/2014; 152(1). DOI:10.1111/ppl.12158 · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Maritime pine (Pinus pinasterAit.) is a widely distributed conifer species in Southwestern Europe and one of the most advanced models for conifer research. In the current work, comprehensive characterization of the maritime pine transcriptome was performed using a combination of two different next-generation sequencing platforms, 454 and Illumina. De novo assembly of the transcriptome provided a catalogue of 26 020 unique transcripts in maritime pine trees and a collection of 9641 full-length cDNAs. Quality of the transcriptome assembly was validated by RT-PCR amplification of selected transcripts for structural and regulatory genes. Transcription factors and enzyme-encoding transcripts were annotated. Furthermore, the available sequencing data permitted the identification of polymorphisms and the establishment of robust single nucleotide polymorphism (SNP) and simple-sequence repeat (SSR) databases for genotyping applications and integration of translational genomics in maritime pine breeding programmes. All our data are freely available at SustainpineDB, the P. pinaster expressional database. Results reported here on the maritime pine transcriptome represent a valuable resource for future basic and applied studies on this ecological and economically important pine species.
[Show abstract][Hide abstract] ABSTRACT: FCBA et l'INRA ont engagé des recherches il y a maintenant près de 20 ans pour développer une méthode de multiplication végétative performante du pin maritime. Ses applications dans le programme d'amélioration génétique seraient multiples, depuis la gestion durable des ressources génétiques jusqu'à la sélection plus efficace des meilleures variétés et leur déploiement facilité dans les plantations. Suite aux développements pionniers chez l'épicéa auxquels FCBA a contribué (années 80, Afocel) et comme chez la plupart des autres conifères, c'est le processus d'embryogenèse somatique à partir de graines immatures couplé à la cryoconservation des embryons somatiques obtenus qui offre actuellement les meilleures perspectives pratiques. Des progrès importants ont été obtenus pour la maîtrise de cette technologie, particulièrement depuis la mise en place en 2004 (et continue depuis) d'une collaboration spécifique sur ce thème entre les équipes " Biotechnologie & Sylviculture Avancée " de FCBA et "Amélioration, Génétique et Physiologie Forestières " de l'INRA. Nous faisons ici un bilan des avancées majeures à différentes étapes de l'embryogenèse somatique jusqu'à la mise en place d'essais au champ en cours d'évaluation. Nous portons également un regard sur les verrous techniques et les contraintes socio-économiques qu'il faudrait lever pour amener cette technologie prometteuse à franchir les portes de l'application pratique.
[Show abstract][Hide abstract] ABSTRACT: Pinus pinaster Ait. is a major conifer in France covering over 1 million ha in intensively managed plantation forests. Together with high productivity (11.8 m3/ha/year), mechanization of marketed harvests (70%) resulted in a significant contribution of this species to the national pulpwood (27.1%, 3.3 million m3) and softwood timber production (24.7%, 5.6 million m3). In France 34 000 workers are employed in this industry (16.5% of the forest sector) with an annual turnover of around 2.5 billion Euros (36% as exports). Breeding programs launched in the early sixties by FCBA and INRA were combined in 1995 into a joined initiative called “Maritime Pine for the Future” that involved all other major forest actors (CPFA, CRPF, ONF). Up to 15% genetic gains were achieved for volume and straightness in first and second generation varieties. Lower genetic gains (10%) are however expected by the third round of selection owing to genetic redundancy within breeding populations. Moreover, maritime pine has a low ability for conventional clonal propagation that would facilitate efficient capture and deployment of the best genetic stocks. The need for an efficient mass propagation system has also dramatically increased because of two recent heavy storms that resulted in a complete clearing of 300 000 ha of forest. To face this challenging task, FCBA and INRA are jointly developing somatic embryogenesis (SE) as a critical enabling technology for efficient elite tree selection, clonal propagation of improved varieties and cryopreservation of both natural and newly obtained genetic resources (Klimaszewska et al. 2007). SE is also providing the plant regeneration system for genetic engineering (Trontin et al. 2007) and serves as a research tool to access the reverse genetics towards marker-aided selection of valuable traits. We will briefly review our major achievements pointing out the current limitations for practical use.
Park, Y.S., Bonga, J.M, Park, S.Y., and Moon, H.K. (eds. 2011), IUFRO Working Party 2.09.02: “Somatic Embryogenesis of Trees: conference on ”Advances in Somatic Embryogenesis of Trees and Its Application for the Future Forests and Plantations, pp100-102; 01/2010
[Show abstract][Hide abstract] ABSTRACT: Some progress has recently been made in establishing a system enabling somatic embryos to be initiated from old elite trees. We report here the first results demonstrating the molecular conformity of somatic embryos initiated from increasingly old Norway spruce (Picea abies (L.) Karst.), as indicated by an analysis of six nuclear microsatellites that showed an extremely high tendency to mutate during in vitro culture. No allelic difference was detected at these loci among plants regenerated from somatic embryos or between the former and mother plants. Moreover, phenotypical data acquired on the same 3- to 9-year-old plants growing in the field sampled for molecular analyses were totally in accord with the results on molecular conformity.