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La embriogénesis es un proceso complejo en las plantas, pero comprenderlo en las coníferas es especialmente crítico no sólo para poder hacer estudios comparativos con angiospermas sino también para producir embriones viables y de calidad con diferentes propósitos. Actualmente falta una visión comparativa global de los genes implicados en la embriogénesis somática y cigótica del pino. En este trabajo presentamos un análisis del transcriptoma en tres etapas del desarrollo de los embriones, identificando procesos biológicos conservados y funciones genéticas activas durante el proceso de embriogénesis somática y cigótica. La mayoria de las diferencias son más significativas a medida que avanza el desarrollo. Cuando comparamos etapas de desasrrollo similares, como es el estadio embrión maduro encontramos 1640 genes sobreexpresados en embriones cigóticos frente a 4814 genes en embriones somáticos. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

International audience

International audience; In Europe, Douglas-fir is a major species for reforestation with increasing demand for its wood. Therefore, adaptation of new varieties to climate change is one challenging question for ongoing breeding programs. Efficient selection and vegetative propagation of improved varieties appeared to be key issues to maintain productivity in plantation forestry (Lelu-Walter et al. 2013). Somatic embryogenesis, coupled with cryopreservation, is a promising clonal propagation system of selected trees that has been successfully improved in Douglas-fir (Reeves et al. 2018; Lelu-Walter et al. 2018). Repetitive somatic embryogenesis from cotyledonary somatic embryos (SEs) has been obtained for two unrelated Douglas-fir genotypes, producing secondary and tertiary embryogenic lines. Each one exhibited significantly higher embryogenic potential for both genotypes compared to primary or secondary lines, respectively (increase of 63 to 727%). The origin of such differences in embryogenic potential is unknown. Our objective was to study changes induced in embryonal masses (EMs) after repetitive somatic embryogenesis at the histo-cytological and molecular levels (LC-MS-based proteomics). Repetitive somatic embryogenesis improved the EM structure by increasing frequency of small, singulated immature SEs together with reducing the size of polyembryogenic centers. Each cycle of embryogenesis induced a modification of the expression of proteins related with biological processes and already known to be involvedin somatic embryogenesis but quite new for EMs i.e.plant development, defense response, metabolism, proteolysis, and stress. The innovative implementation of protein networks in our proteomic analyses had been very conclusive. The networks revealed a global down or up pattern of protein expression after the first or second cycle of somatic embryogenesis, respectively. For both patterns, interactions with different plant growth signaling molecules (flavonoids and associated compounds, jasmonic acid, ABA, auxin, salicylic acid, lignin) were predominant. It is shown that cells have the ability to use different protein regulatory pathways to result in increased embryogenic potential (cotyledonary SE production). In Douglas-fir and in conifers, this is the first report describing cellular and molecular changes in EMs obtained after two successive cycles of repetitive somatic embryogenesis (Gautier et al. 2018). The results contribute to a better understanding of the cytological and proteomic changes associated with to enhanced embryogenic potential of secondary and tertiary embryogenic lines.

La fenilalanina es un aminoácido esencial para la síntesis de proteínas pero también un precursor de una gran variedad de compuestos del metabolismo secundario que son esenciales para el crecimiento, desarrollo y defensa de las plantas. La biosíntesis de la pared celular secundaria que tiene lugar durante la formación de madera en árboles implica la biosíntesis masiva de lignina, un polímero que no contiene nitrógeno pero que deriva metabólicamente de la fenilalanina. Por lo tanto, estas plantas requieren una coordinación metabólica precisa entre la biosíntesis de fenilalanina y la biosíntesis de lignina para asegurar su desarrollo y crecimiento anual. En este estudio, hemos encontrado que la enzima arogenato deshidratasa, que cataliza el último paso en la ruta biosintética de la fenilalanina en las plantas, se regula transcripcionalmente a través de la interacción directa con el factor de transcripción PpMYB8. El análisis transcripcional de plantas de pino marítimo silenciadas para PpMyb8 sugiere que este factor de transcripción está directamente involucrado en la biogénesis de la pared celular secundaria y en los procesos de muerte celular. En conjunto, estos resultados indican que un único factor de transcripción coordina la biosíntesis de fenilalanina y la acumulación de lignina durante la formación de madera en las coníferas. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Douglas-fir (Pseudotsuga menziesii (Mirb) Franco) is a native conifer from the Pacific North-West of the US and Canada, and is one of the most important timber species used in the world. Douglas-fir is highly productive in a range of climatic conditions and has valuable wood properties (quality) as well as a strong tolerance to diseases and insects. In Europe, Douglas-fir is a major species for reforestation with increasing demand for its wood. Adaptation of new varieties to climate change and associated stresses is one challenging question for ongoing breeding programs. Efficient selection and vegetative propagation of improved varieties are key issues to maintain productivity in plantation forestry (Lelu-Walter et al. 2013), however, as in many other conifers, early maturation is preventing clonal forestry through conventional multiplication methods in Douglas-fir (Bastien et al. 2013). Somatic embryogenesis from immature seeds, coupled with cryopreservation, is a promising retroactive clonal propagation system of selected trees. This technology has been developed for an increasing number of conifer species (Klimaszewska et al. 2016). Excluding patents, there are only a few published studies on Douglas-fir somatic embryogenesis (Durzan and Gupta 1987; Pullman et al. 2005, 2009). One recurrent problem is the sustainable multiplication of initiated embryogenic material, i.e., embryonal masses (EMs). Yellowish, non-embryogenic cells (NEC), which are interspersed with EM, is frequently observed during this process. In this work, we describe EM morphology and cytology (light and confocal microscopy) of different embryogenic lines. A suggested method to reduce the formation of NEC and sustain EM proliferation is presented.

International audience

International audience; Douglas-fir (Pseudotsuga menziesii (Mirb) Franco) is a native conifer from the Pacific North-West of the US and Canada, and is one of the most important timber species used in the world with both high productivity in a range of climatic conditions and highly valuable wood properties (quality) as well as strong tolerance to diseases and insects. In Europe, Douglas-fir is a major species for reforestation with increasing demand for its wood. Therefore, adaptation of new varieties to climate change and associated stresses is one challenging question for ongoing breeding programs. Efficient selection and vegetative propagation of improved varieties appeared to be key issues to maintain productivity in plantation forestry (Lelu-Walter et al. 2013). However, as in many other conifers, early maturation is preventing clonal forestry through conventional multiplication methods in Douglas-fir (Bastien et al. 2013). Somatic embryogenesis from immature seeds, coupled with cryopreservation, is a promising retroactive clonal propagation system of selected trees, currently developed for an increasing number of conifer species (Klimaszewska et al. 2016). Excluding patents, there are only a few published studies on Douglas-fir somatic embryogenesis. One recurrent problem is the sustainable multiplication of initiated embryogenic material, i.e. embryonal masses (EMs). Currently, EMs obtained after initiation are subcultured every two weeks in clumps on proliferation medium. Yellowish, nonembryogenic callus (NEC), wich is interspersed with EM, is frequently observed during this process. In this work we used high-resolution optical microscopy for cytological observation of three embryogenic lines initiated in 2012 (from different genotypes: D1, D2, D3) that were previously classified according to their macro-morphology as either “pure”, white-translucent EM as typically described in conifer species (D1, D2) or as a mixture of EMs and NEC (D3). Using confocal microscopy with FDA/PI staining it was shown that EMs from a “pure” line (D2) are apparently interspersed with viable, persistent non-embryogenic cells of unknown origin. In addition, dead cells were observed in embryo suspensors from embryogenic lines suggesting actively occurring apoptotic and autophagic programmed cell death (PCD) that is required for normal embryo development. To tentatively reduce the occurrence of non-embryogenic cells, it is necessary to frequently subculture the EMs (each week), to vigorously dissociate them in liquid medium before transferring onto filter paper as a thin cell layer (Reeves et al. submitted).

International audience; Current somatic embryogenesis (SE) protocols for many pine species remain suboptimal for the purpose of reforestation. Some of the bottlenecks include losses of lines through the SE process due to lack of continued proliferation, poor embryo maturation or poor conversion of somatic embryos to ex vitro conditions. There is often significant variation among genotypes with respect to productivity and SE protocol improvements do not always benefit all genotypes. There is often the desire to propagate specific cell lines, for example from top-ranked families. Selected lines may show desirable characteristics such as disease resistance or a high ability to be genetically engineered. There is also a need to propagate cell lines from species that are in general recalcitrant to somatic embryogenesis (e.g. Pinus densiflora). Cell lines from hybrid crosses, where there can be incompatibility issues between parent species, also present several challenges. In particular there are no protocols available for these new hybrid crosses which are often less responsive to all steps in the SE process. An example of this recalcitrance could be a cell line which is highly transformable but only produces a limited number of good quality mature somatic embryos. This makes direct planting of somatic embryos to ex vitro conditions an unviable option. If the desired cell line will not respond positively to modification of the standard SE protocols or media for the species then the options for bulking up this material are limited. A hybrid tissue culture system combining germinated somatic embryos with organogenesis allows the flexibility to work with these desirable cell lines and allows the best aspects of the component propagation methods to be exploited. In conclusion, we have a propagation protocol that takes advantage of the benefits of SE, which include cryopreservation to ensure juvenility while field testing takes place and the potential for genetic engineering. These features combined with organogenesis, which provides the advantages of increased early multiplication, uniformity of shoots and high rooting and conversion to planting stock/stoolbeds is a winning strategy for some conifer species. This talk will highlight where Scion has used this hybrid system to circumvent issues around certain protocol stages, cell lines and conifer species.

chapitre en open access; International audience; The genus Pinus includes several species that are economically important and planted outside their natural ranges as plantation species. Somatic embryogenesis (SE), a biotechnological tool for mass propagation of pines, has been reported for many species but only from seed embryos. Cloning the individual adult trees, through SE from vegetative explants, could potentially benefit the forest industry in that only trees with elite characteristics would be planted commercially. The attributes of conifer trees may only be evaluated after many years of growth and often not until the reproductive growth phase. This chapter describes a concerted effort by several research teams, in five countries, to initiate SE in primordial shoot explants of six pine species, each commercially important in its respective country. In spite of the multiyear experimentations, SE was induced in only one species (Pinus sylvestris), but embryogenic lines showed some instability at microsatellite loci and the somatic embryos did not germinate. Some cell lines initiated in different species showed embryogenic-like characteristics at the microscopic level. Expression of embryogenesis specific genes (LEC1/CHAP3A, WOX2, VP1) was detected in such calli/cell aggregates of all three tested pine species (including those with embryogenic-like characteristics) even when the presence of early somatic embryos could not be confirmed. Overall, the results presented in this chapter are indicative of the existing challenges in propagation of adult pines as in other conifers.

National audience; Maritime pine is a major forest tree in southern Europe for sustainable delivery of bio-based products through advanced plantation forestry of improved varieties. Early selection is needed to reduce breeding cycle and accelerate variety deployment in the context of climate change. Both gene(s) and genome-wide information should provide opportunities for predictive, marker-assisted selection. Reverse genetics, defined as ectopic expression or silencing of candidate genes can be useful for functional dissection of traits of interest. Functional genomics of 9 transcription factor (TF) genes associated to growth and wood formation (MYB1,8,14,20,23, DOF5, MADBOX4, NACx, NACataf) was initiated in maritime pine through genetic transformation of embryogenic tissue. Twelve TF constructs designed for constitutive overexpression (OE) or silencing (RNAi) were studied: 6 constructs (batch 1) from previous projects (MYB1-RNAi, MYB8-OE/RNAi, MYB14-RNAi, DOF5-OE/RNAi) as well as 3 constructs for putative gene targets of MYB (CAD-RNAi) and DOF5 (GS1a-OE, GS2-OE); and 6 constructs (batch 2) obtained during ProCoGen (MYB23-OE, MYB20-OE, MADBOX4-OE, NACx-OE/RNAi, NACataf-OE). Phosphinothricin-resistant lines could be cryopreserved for all but one (MYB20-OE) constructs. Transformation rate was estimated in the range 9.6-22.0 (OE) or 0.4-18.0 (RNAi) transgenic lines per gram embryogenic tissue (batch 1). Somatic embryos were obtained from 1-3 lines per construct (25 lines in total) and successfully converted to plants (batch 1). Acclimatization rates were similar to controls and transgenic plants were confirmed for most lines. Putative adverse effect of MYB14-RNAi, DOF5-RNAi, and MYB8-OE on transformation rate was observed. DOF5-RNAi apparently stimulated germination rate. Plant growth data and morphology are available for constructs from batch 1 after up to 12 (MYB1-RNAi, MYB8-OE/RNAi, DOF5-OE/RNAi, GS2-OE, GS1a-OE) or 42 months (MYB14-RNAi, CAD-RNAi) growth. Both transgene copy number and targeted gene expression data are available from transgenic plants obtained from MYB14-RNAi and CAD-RNAi lines. Transgenics and controls from batch 1 were sampled at age 16 (MYB1-RNAi, MYB8-OE/RNAi, DOF5-OE/RNAi) or 70 months (MYB14-RNAi, CAD-RNAi). Molecular characterization of MYB1,8,14 and DOF5 plant material has been performed by qPCR and transcriptomic analysis using microarray is underway. Wood analyses have been initiated for MYB14- and CAD-RNAi plants (in progress).

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 zygotic 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.

The potential to use micropropagation protocols in combination with a nursery production system via stool beds is attractive. Somatic embryogenesis protocols would amplify high value control-pollinated seed and organogenesis of the resulting mature somatic embryos (SE) would provide a large number of uniform shoots for initial production of stool beds. SE material held in liquid nitrogen could then be used to provide a supply of juvenile stool beds over a number of years. Successful results have been obtained with initiation and proliferation of Douglas-fir embryogenic cultures at Scion. These protocols have been developed using modified Pinus radiata media and methods and provide Scion with protocols for the micropropagation of Douglas-fir. Mature somatic embryos (from 5 cell lines) had their bases removed and were placed on Pinus radiata organogenic media (LPch) to establish shoot cultures. Shoots were sub-cultured every 4-6 weeks, transferring to jars when elongated. Whenever possible, stem segments were isolated from the elongated shoots and transferred to petri dishes for multiplication. Multiplication data was collected for 30 weeks. Thirty organogenic shoots from each of the five SE cell lines were used to test four different pre-rooting treatments. Shoots were transferred to the nursery propagation house after 11 days. The results of these pre-rooting treatments on the ability of the shoots to produce roots will be presented and future work discussed.

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International audience; Development of clonal propagation method, such as somatic embryogenesis (SE) has potentially numerous applications such as the production of a large number of genetically improved plants. However it is necessary to optimise somatic embryo development, which remains difficult in pine species. Since the early protocols of SE were developed for spruce species (Picea), it was assumed that they would be applicable to pines; however, it became apparent that pines were less responsive. One limitation to large-scale propagation of majority of commercial pine species through SE is the lack or low somatic embryo maturation efficiency, which result in the inability to capture certain families. This has caused great concerns among breeders and foresters of potential adverse selection pressure. Current maturation protocols lead to a development of mature somatic embryos that morphologically resemble zygotic embryos. These somatic embryos are harvested after arbitrarily chosen periods of time and are germinated and further grown in a greenhouse. This procedure only gives an indication that the maturation was successful if produced somatic embryos were able to convert to plants. Such an empirical approach does not give any information of the quality of somatic embryos with respect to storage reserves accumulation nor does it gives information on the optimal time for harvesting to achieve maximal plant conversion rates. Therefore, there is a need to develop markers that could be used for quality control of different batches of somatic embryos that are matured, or when different maturation protocols are applied. Storage protein accumulation has been followed and the identity of the proteins accumulated by the somatic embryos has been tested by 2D gel electrophoresis. Final objective is to have a better understanding of the maturation of Pinus pinaster. The description of SE would contribute to optimise the maturation process and the in vitro production of plants.

Maritime pine is extensively used in south-western Europe for lignocellulose production. Increasing demand for wood from this pine species requires efficient production of improved varieties and this is currently achieved only through seed orchards management. As in most long-lived tree species, variety design and deployment would greatly beneficiate from setting up an efficient vegetative propagation system of selected seed resources. Somatic embryogenesis is a promising clonal propagation system in most conifers and significant progress has been made in recent years for maritime pine (reviewed in Klimaszewska et al. 2007). However the « true » maturity of harvested cotyledonary somatic embryos (SE) is still a debatable question. Cotyledonary somatic embryos are currently selected at the end of the maturation phase based on morphological criteria. Embryo maturation is a crucial step giving rise to the establishment of reserve compounds including specific storage proteins (Teyssier et al., 2011) that will ensure optimal germination and subsequent in situ plantlet development. A 2D-PAGE proteomic approach has been developed at INRA of Orléans to define the protein status of developing SE in reference with zygotic embryos (ZE). We aimed at contributing for a better understanding of the processes acting during the maturation phase of SE (part 1). As a first step towards this goal this work is presenting water content and percentage of dry mass in maturing SE vs. ZE (part 2) and our first results in proteomic analysis of SE development (part 3). This new knowledge is expected to ultimately results in refining our current methodology for practical application of somatic embryogenesis in maritime pine.

National audience; In order to get insights into the physiological mechanism underlying the development of somatic embryos of the maritime pine (Pinus Pinaster), experiments at different stages of embryos maturation and different water availability conditions were undertaken to identify protein markers. To our knowledge, this is the first proteomic investigation on somatic embryosof Pinus pinaster.

International audience; Initiation of somatic embryogenesis (SE) from immature excised zygotic embryos (IZE) in maritime pine was originally developed at FCBA using a modified DCR basal formulation (mDCR) with the 2,4-D auxin (13.5 μM) and the BA cytokinin (2.2 μM) as plant growth regulators (PGRs). In the frame of an international collaboration (initiation campaigns 2003-2004) we discovered that a modified Litvay basal medium (mLV) supplemented with the phenylurea cytokinin CPPU (4.04 μM) produced a significant increase in mean initiation rate (77 vs. 34%, Park et al. 2006, PCTOC 86: 87-101). This result was confirmed and strengthened during subsequent 2005-2008 initiation campaigns. Up to 92% mean initiation rate (2005 results) were obtained using the mLV/CPPU (4.04 μM) medium with quite moderate variation between years (62-92%, 4-5 families investigated per year, 200-300 IZE/family). The optimal CPPU concentration was studied and we concluded that up to a fourfold reduction of the original dose (1 μM) could be used without significant effect on initiation rate. Similar conclusions were drawn by reducing the initiation culture time of IZE on mLV/CPPU (4 μM) from 8 weeks to only one week. Interestingly initiation rates remained high (47-55%) when initiation media were deprived of CPPU, thus confirming the high interest of mLV for SE initiation in maritime pine. Substituting CPPU (4.04 μM) for the more conventional 2,4-D (9.04 μM) and BA (4.44 μM) PGRs in combination with 10 or 30 g/l sucrose significantly reduced initiation rates. mLV/CPPU (1 μM) was proposed as the optimal initiation medium at FCBA laboratory.

Advances in conifer biotechnology offer new opportunities in the field of vegetative propagation and genetic engineering. Development of clonal propagation methods, especially somatic embryogenesis (SE), has numerous potential applications. Owing to its efficiency in plant regeneration, coupled with the ease of long-term storage in liquid nitrogen, SE became an indispensable tool for accelerating the development of tree varieties and deployment. Early SE protocols were developed for Picea species, however, when applied to Pinus species, especially at the somatic embryo maturation stage, they were unsuccessful. It became apparent that pines required more research and development to bring the SE biotechnology to its potential. This review emphasizes the most recent progress made in SE and cryopreservation in the genus Pinus, focusing on major plantation and forest species in Europe (Pinus pinaster), New Zealand, Australia, and South America (P. radiata), as well as North America (P. taeda and P. strobus). Much consideration is also given to applying SE in pine improvement and deployment strategies in multivarietal forestry.

Advances in conifer tissue culture and genetic transformation offer new opportunities in the field of genetic engineering. Genetic transformation is an important tool for breeders because it allows them to introduce valuable genes that might otherwise be difficult to integrate into elite genotypes. Combined with mass vegetative propagation, such as somatic embryogenesis, regeneration of transgenic conifers could result in accelerated tree improvement. Genetic transformation requires the development of an efficient gene delivery system. Extensive work has been done in Pinus spp. to improve transformation methods, either by DNA-coated particle bombardment or by co-culture with Agrobacterium tumefaciens. This review describes the most recent progress made in genetic transformation in the genus Pinus, with emphasis on four important forest and plantation species grown in Europe (P. pinaster), New Zealand, Australia and South America (P. radiata), and North America (P. taeda and P. strobus). The biosafety issues associated with potential deployment of transgenic pine varieties in commercial forestry are highlighted.