[Show abstract][Hide abstract] ABSTRACT: Significant progress has been made in the development of maritime pine somatic embryogenesis but there are still technical issues precluding full integration of this powerful vegetative propagation system into the French breeding programme. Maritime pine somatic embryos (SEs) 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 the transcriptome (Illumina RNA sequencing) and proteome (2D-SDS-PAGE with MS identification) of immature SEs, cultured on either high (9 gl-1 , 9G) or low (4 gl-1 , 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 SE development. Under conditions unfavorable for SE maturation (4G) both transcriptomic and proteomic profiling indicate enhanced glycoly-sis leading to proliferation of embryonal masses (EMs) which may be antagonistic to SE maturation. Under favorable conditions (9G), we observed adaptive, ABA-mediated molecular and physiological responses to reduced water availability resulting in early transition of EMs from proliferation to the SE developmental pathway (indicated by active protein synthesis, and overexpression of proteins involved in cell division, em-bryogenesis and starch synthesis). Specific pathways (synthesis of protective secondary metabolites, regulation of oxidative stress) are also activated, apparently to overcome constraints due to culture conditions. This is the first study on the early molecular mechanisms involved in somatic embryogenesis of pine following an increase in gellan gum concentration in the maturation medium, and it is also the first report combining transcriptomic and proteomic data analysis during somatic embryogenesis in conifers. We have found novel candidate predictive markers for conifer SE development (germin-like protein and ubiquitin-protein ligase) as well as for adaptive responses (protein phosphatase 2C), which may facilitate practical application of the knowledge gained in this study to monitor early responses of embryogenic tissue to maturation conditions.
[Show abstract][Hide abstract] ABSTRACT: In maritime pine, improved protocols are now available for the whole somatic embryogenesis process i.e. from initiation of embryogenic tissue to somatic plant regeneration. However, field trials established in France from somatic plant material have consistently revealed that somatic embryos (SEs) developed at a lower initial growth rate compared to control seedlings. A better understanding of SEs maturation is therefore required in order to produce high-quality, vigorous somatic plants. SEs are currently matured for 12 weeks to reach the cotyledonary stage before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the harvesting date is still determined from 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 significant difference could be found between collection dates, suggesting that SE harvesting after 12 weeks maturation is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygo-tic embryos (ZEs). Using hierarchical ascendant cluster analysis, we demonstrated that cotyledonary SEs matured for 12 weeks are most similar to fresh cotyledonary ZEs sampled from late July to early August (as indicated by dry weight, water content, sucrose, RFOs content, RFO/Sucrose ratio) or at any time up to October (with respect to total protein). Both types of embryo exhibited similar carbohydrate and protein content 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 (LEA) 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. Of these, 18 belonged to five large families of proteins including five HSPs, four LEAs and two other stress-related proteins (aldose reductase, 6-phosphogluconate dehydrogenase), five storage proteins and two proteins involved in purine metabolism (adenosine kinase 2, SAM synthase). 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. Acknowledgements: This work was supported by grants from the French " Conseil Régional de la Région Centre " (EMBRYOME project, contract 33639) and " Conseil Régional de la Région Aquitaine " (EM-BRYO2011 project, contract 09012579-045). Some equipment used at FCBA to get results presented in this work was funded by ANR-10-EQPX-16 XYLOFOREST. We gratefully acknowledge Gilles Bruneau, Francis Canlet, Sandrine Debille, Karine Durandeau, Séverine Quoniou and Françoise Wan-Fung (FCBA) for their valuable contribution to the collection of somatic and zygotic embryos and Pierre Alazard (FCBA) for cone sampling in the field. We acknowledge Marlène Bailly for her technical assistance in electropho-resis.
[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.
Full-text · Article · Jan 2014 · Physiologia Plantarum
[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.
Full-text · Article · Nov 2013 · Plant Biotechnology Journal
[Show abstract][Hide abstract] ABSTRACT: Maritime pine is a major species for the French industry covering over 1 million hectares with a total production of 9 million m3 representing about 25% of total pulpwood and softwood timber and an annual turnover of 2.5 billion €. Most of this production is localized in the Aquitania forest in SouthWestern France. As the main intensively managed conifer in France, maritime pine is subjected to advanced forestry and genetic breeding since the early sixties. After 40 years research, a 40% cumulated genetic gain is expected by the third round of selection; but there are also limitations to improvement progress, i.e. quite long generation time, large genetic loads and high genetic redundancy within breeding population. Sudden and drastic changes in market value and environmental constraints in recent years (2 heavy storms during the last decade, increased drought and biotic stress) would need a significant paradigm shift in current breeding technology to deliver suitable tested tree varieties in plantation forestry (i.e. multivarietal forestry targeting a wide range of end-products through various silvicultural regimes). Field comparison of vegetative propagules is a key towards individual selection and efficient capture of the best genetic stocks. Clonal propagation is also required for scaling up production of improved varieties. Somatic embryogenesis (SE) is considered as the key technology to fulfil such requirements in maritime pine. As SE initiation from mature trees is still challenging in pine and other conifers, we are developing in maritime pine the classical SE approach for postponed propagation of tested trees involving SE initiation from immature zygotic embryos and stable cryopreservation of juvenile embryogenic tissue. Genotype captures at both SE initiation and maturation steps are key issues for successful implementation of this technology. The species, characterized by low vegetative propagation ability (cutting production is very difficult and particularly expensive, at least in Europe, compared to some other pine species). At the initiation step, family effect was confirmed to be highly significant. However with a mean genotype capture within family of 77% the variation among genetic backgrounds is now established within acceptable limits and has huge practical implications for our breeding program. The maturation step is currently more problematic but recent progress resulted in about 40% genotype capture. Major findings will be synthesized from 10 years maturation experiments at both FCBA and INRA.
[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.
[Show abstract][Hide abstract] ABSTRACT: Growth of unselected SE lines was compared to seedling material from the same genetic A view of 10-15 year-old French Norway spruce somatic embryo field trials (AFOCEL program). FCBA is the french technological institute of the whole forest-wood-paper & furniture chain. It was funded in 2007 as the merger of AFOCEL and CTBA. AFOCEL was one of the pioneers of conifer Somatic Embryogenesis (SE) during the 1980's. Research was initially focussed on Norway spruce to understand the basic events associated with SE and to set up methods, regarding particularly rejuvenation (SE from increasingly aged seedlings and tree). Due to the drop of commercial demand for Norway spruce seeds and plants on the French market, the Norway spruce SE program was terminated in 1999 as the same time as the breeding activity on this species, redirecting efforts towards Pine. At this time, although hundreds of thousands of plants were produced on experimental material, only a few field trials have been established. Here we present an overview of three of these trials. Growth of unselected SE lines was compared to seedling material from the same genetic provenances on the field over nine to twelve years. Although unselected clones were used, the growth of SE plant material was good. In two out of three trials, SE material perform as well as the corresponding seedlot (one year younger). The third trial, planted with material from earlier plant batches, shows lower performance of SE material. Complementary experiments, including additional field trials, demonstrated that this lower performance was due to suboptimal ex vitro management. Indeed, the slower initial growth of emblings necessitated adaptation of plant management to get good performance. Acknowledgement : The establishment of these filed tests was partly funded by EU through FP5 contract n° FAIR950873
[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.
[Show abstract][Hide abstract] ABSTRACT: Pinus pinaster is one of the most important species for the French wood and paper industries. It covers more than 1 million ha. Strong efforts are focused to genetically improve the species and make available more efficient sylviculture practices. Somatic embryogenesis is presently considered as part of the breeding programs. This paper presents a summary of AFOCEL results on initiation of somatic embryos clones from immature seeds since the early experiments in 1988 until 2000 with a focus on unpublished data (year after 1994). Immature embryos from controlled pollinated elite trees have been harvested during the summer time, taking into account the competence window defined in late 80's. The initiation rate of embryonal suspensor masses (ESM) was strictly scored at the level of stabilized proliferating material to avoid artificial overestimation. It was revealed to be highly variable both among crosses and among years for the same cross despite harvesting of plant material at the same time of the year and using only embryo at the precise developmental stage defined as optimal. Medium comparison during rapidly concluded to the superiority of DCR, which allows more regular and higher results on average despite not being the best in each particular case. In addition to precluding the checking of the developmental stage of each zygotic embryo (ZE) put in culture, presence of the megagametophyte tends to give us a lower initiation rate. No correlation between ZE development and temperature data could be observed, especially during the last years that were significantly warmer in the Landes area where are located the seed orchards. No strict parental effect could be seen but availability of cones of a particular cross is difficult to obtain regularly each year, making very hazardous the possibility to replicate experiments. The results merely suggested a physiological influence of the mother tree rather than a strictly mono-or bi-parental genetic effect. When weather and cone characteristics are good, initiation rates average 45%, reaching 75% for some crosses with the recovery of ESM from each family.
[Show abstract][Hide abstract] ABSTRACT: Somatic embryogenesis is quite well mastered for spruce species. At least in western Europe, the most limiting factors for it's practical use are the high manpower requirement leading to a production cost somewhat higher than that of standard seedlings and the lack of interest for clonal propagation (increased by the confusion between clone and GMO) of most foresters. We designed an experiment to situate the security limit to warrant reliable and efficient plant regeneration from somatic embryos culture in terms of subculture number or total time of continuous multiplication. A large amount of 4 different performing lines where cryopreserved after a limited number of subcultures while they where maintained in continuous culture. Cryovials where thawed along a 100 weeks period after increasing amount of time and plant material reactivated. At each of the 10 time points, stably reproliferating cultures where matured in parallel with unfrozen and, if already present, previously thawed ones. Except for one line showing a very fast degeneration, no long-term stable trend could be detected at the level of plant regeneration ability. Plant production was either increasing with subculture number for sometimes quite long period (40 weeks for example). If maturation yield varied significantly according to the multiplication and maturation systems (solid or liquid culture, spreading or not ESM in thin layer for maturation...), the evolution of mature embryo numbers where quite parallel. The line showing a very fast collapse of maturation ability (down to almost zero in 3 months) had a microscopical aspect typical of B lines (after the terminology of the team of S. von Arnold) concomitant with a granular aspect with fast draining and drying of ESM sedimented from suspension culture. The best results were always obtained with material matured as a thin layer on filter paper laid on the top of solid medium from a proliferation done either on solid medium or in suspension. At the level of sanitary safety (susceptibility towards infection) as well as at the level of cost of long term maintenance of a minimal amount of plant material, the solid proliferation prove to be the best and the cheapest. In conclusion, we choose to adapt the time of continuous maintenance in culture to the speed of the maturation ability lost. Each newly initiated line is cryopreserved as soon as possible and then its the relative speed to loose the potential of plant production is estimated on maturation test replicated at least 5 times along several months. The time limit is thus know for fast decaying material while more persistent lines are not maintained for more than one year but this limit was chosen considering sanitary risks overall.
[Show abstract][Hide abstract] ABSTRACT: While somatic embryogenesis of spruce species is pretty well mastered by numerous labs, some teams still have difficulties to produce well growing plants. One of the factors explaining such a situation is the management of ex vitro growth and particularly the period of time just following the transfer of plants from the lab to the greenhouse. After having improved all classical parameters of greenhouse plant management, we still had troubles of precocious growth arrest of terminal buds. This blocking was irreversible in most of the case, leading to abnormal morphologies. The solution to this problem was found empirically in early the 90's at AFOCEL, as it seems to have been the case in many other labs at the same time or several years later. It consists in using extended photoperiod for some weeks until the terminal bud will have gone through the critical stage of growth restart after the stress of weaning. As the things worked fine, we don't assign a high priority to the proper scientific validation of this technical point. We are now presenting detailed experimental results on several clones showing precisely what is the impact of different light regime on the growth of young emblings along their first months outside the lab.
[Show abstract][Hide abstract] ABSTRACT: There are more than 4 million hectares of Pinus pinaster forest in mediterranean Europe. Among these countries, somatic embryogenesis of this species has been studied for years mainly in France (AFOCEL and INRA), while some projects later in other countries. Australia is also becoming an important pole with the long term objective to reach at least 500,000 ha. They started also to work on somatic embryogenesis a few years ago. In France, while work started in late heighties, some difficulties still persist. At the starting point of the process, the initiation rate from controlled cross immature seeds is high and proliferation is efficient, leading to a high rate of stabilized immature embryos, proliferating in colonies called embryonal-suspensor masses (ESMs). Cryopreservation is routinely done with a high success, leading to large cryobank establishment allowing to secure a significant part of the genetic variability of breeding populations, allowing to integrate somatic embryogenesis in breeding programs like most of the big forest companies are now very actively doing with their species of interest. Like with other conifers, ABA is not sufficient to trigger the differentiation of immature into cotyledonary embryos (stage 3). The development of immature embryos into cotyledonary embryos is one of the major problems preventing the large deployment of somatic embryogenesis in Pinus pinaster. But significant progress were made during the last years. The influence on the maturation of maritime pine somatic embryos of carbon sources and PEG combined with increasing concentration of gellan gum was tested. The effect of the 2 factors vary widely between lines. Some lines showed a striking response to PEG treatment, which limited the ESM proliferation and enhanced the maturation rate. Conversely, proliferation was stimulated by PEG in other lines without subsequent improvement of the maturation rate. The use of high concentration of gellan gum (0.9%) improved the maturation of all ESM lines. It was concluded that the more efficient culture medium to recover cotyledonary embryos from all lines was supplemented with sucrose at 6% (w/v) and gellan gum at 0.9% (w/v) without PEG. It apperas also that the maturation performance is highly influenced by sampling modalities; the outer part of the ESM yielded more cotyledonary embryos than the inner part or the whole colony. ESM lines showing several stage 1 embryos at the periphery (spiky) were more productive than those for which stage 1 embryos were rarely visible (smooth). Impact of gelling and carbohydrate on plant regeneration will be also discussed together with the effect of genetic component.