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Sorghum is a crop widely cultivated in southern countries (Africa, Latin America, India) for human and animal consumption. It is also grown in northern countries for animal consumption and increasingly used for energy and biomaterials. These uses often rely on vegetative biomass valorization. Most of this biomass accumulates in secondary cell wall (SCW) of the stems as celluloses and xylans, which are tightly linked to lignins. Being able to monitor precisely the composition of SCW in sorghum can provide a major leverage to optimize its different uses. Understanding how SCW is deposited and regulated would allow breeding programs to develop varieties fitting the expectations of the different value chains. The identification of genes involved in SCW development in sorghum will contribute to improve our ability to optimize its composition and structure. Transcription factors (TF) are key targets because they are often responsible for fine-tuning biological processes. Moreover, they allow deciphering the downstream network. MYB and NAC TF families are already known to be involved in SCW regulation in other model species, but their roles still need to be addressed in sorghum. For this study, we carried out comparative genomics and co-expression network analysis to highlight NAC and MYB genes that are most likely to be involved in SCW regulation in sorghum. Based on these candidates, functional validation experiments are on-going. Firstly, the comparative genomics analysis identified 96 NAC and 170 MYB genes in sorghum. 34 NAC and MYB genes were recognized as orthologs of master regulators of SCW in other species. Moreover, phylogenetic dissimilarities within both families between monocotyledons and dicotyledons pointed out potential differences in SCW regulation between these two groups of angiosperms. Secondly, using transcriptomic data from developing sorghum internodes, we built co- expression networks. Using orthologs of previously identified TF as baits, we were able to generate genes lists potentially involved in the same process of SCW biosynthesis. Genes co- expressed with bait TF showed enrichment in functional categories (GO terms) linked to SCW. They also revealed differences in enriched categories for TF related to primary cell walls or specific compounds of SCW. Some whole co-expression network groups are also enriched in SCW linked categories. It is likely that there are new important SCW involved genes within these candidate gene lists. Taking advantage of genome wide association results, we detected overlaps between the newly identified list of genes and genomic regions controlling stem composition. Some of the sorghum TF roles are being validated by targeted transgenesis (CRISPR-Cas9 and overexpression). In the future, these results will contribute to the management of the diversity of SCW key genes within selection programs for sorghum stems quality improvement.

The mantled somaclonal variation of oil palm (Elaeis guineensis Jacq.) is morphologically similar to mutants defective in MADS-box genes of the B-class. Indeed in such mutants male floral organs are converted into female ones and this can be observed in oil palm flowers of both sexes. The detrimental consequences of somaclonal variation on fruit set then oil yields and its unpredictable incidence in clonal progenies have prompted the search for early detection markers. More generally, a better understanding of the molecular mechanisms underlying the onset and the maintenance of this variant phenotype is needed. The mantledsomaclonal variation is observed only in clonal populations generated through somatic embryogenesis at a large commercial stage. Thus, because of the nature of the variation, research strategy must involve two parallel approaches aimed at studying simultaneously the in vitro stages of clonal palms production (the flask) and the in plantadevelopment of the reproductive organs which are affected by the phenotype (the flower). On both materials a novel combination of epigenetics approaches, high-throughput sequencing technologies and bioinformatics analyses now provide new insights into a complex developmental phenomenon. Our research work also paves the way for innovation and development oriented towards end-users in the palm oil industry. (Texte intégral)

The mantled somaclonal variation of oil palm (Elaeis guineensis Jacq.) is morphologically similar to B-class MADS-box genes mutants: floral organs undergo a male-to-female homeotic conversion in flowers of both sexes. The negative economic consequences of this somaclonal variation on oil yields, as well as its variable incidence in clonal progenies, have made it necessary to understand the molecular mechanisms responsible for the variant phenotype. We have recently demonstrated that the splicing of EgDEF1, the APETALA3ortholog that is believed to be involved in the mantled floral phenotype, is significantly altered in variant flowers with respect to normal ones, resulting in the over-accumulation of a shorter, and potentially defective, transcript isoform (Jaligot et al., 2014). We are now attempting to determine through In Situ Hybridization whether these alternative transcripts could indeed compete within the same tissues. Also, because EgDEF1is inserted in a region of the oil palm genome that is rich in Transposable Elements (TEs), we are investigating the possibility that TEs might play a role in the onset of the variation in the course of the somatic embryogenesis process. (Texte intégral)

The mantled variation of oil palm (Elaeis guineensis) involves a significant deficit in genome-wide methylation as well as the mis-expression of a variety of genes affecting both the in vitro-propagated material from which the palms originate and the adult regenerants (for a review see [1]). This reversible floral phenotype is morphologically similar to B-class MADS-box mutants: indeed, the abnormal flower displays an homeotic conversion of male floral organs into female ones [2]. In order to investigate the role of epigenetic processes in the transcriptional regulation of EgDEF1, the ortholog of APETALA3/DEFICIENS in the oil palm genome, we have studied the DNA methylation pattern of this gene concomitantly with its transcript production in the developing inflorescence. The exploration of the genomic region surrounding the gene led to the isolation of several retroelements which were also examined. Although our results do not point to a direct implication of DNA methylation in the regulation of the EgDEF1 gene, they raise exciting new questions about the possible mechanisms underlying the variant phenotype. (Texte intégral)

The mantled floral phenotype which occurs in somatic embryo-derived oil palms (Elaeis guineensis Jacq.) shows detrimental consequences on the large scale production of clonal planting material for this strategic oil crop. Indeed, mantled somaclonal variants display a feminization of male floral organs which bears a major threat on the formation of oil-producing fruits. The unpredictability of such variation warrants the search for molecular markers for an early detection. In parallel, the mantled phenotype provides a challenging puzzle to the researcher since it is both reminiscent of floral abnormalities governed by MADS Box genes in model plants and susceptible to provide insights into the particulars of sex differentiation and flower organogenesis in Palms species. Since a genome-wide deficit in DNA methylation has been demonstrated in mantled tissues and since gene expression differs substantially with respect to true-to-type material, it is now widely accepted that this unstable variant phenotype is correlated with (and likely caused by) the disturbance of epigenetic mechanisms during the in vitro micropropagation process, which involves a series of differentiation/dedifferentiation phases. Our talk provides an update on ongoing research work aimed at deciphering the role of epigenetic regulations in the floral development of the oil palm. (Texte integral)

The oil palm (Elaeis guineensis Jacq.) is the first world source of vegetable fats, and as such it is generating a great amount of research efforts. In particular, understanding the molecular mechanisms underlying the setting up of the reproductive organs and their consecutive development in flowers and fruits appears essential in the perspective of improving oil production. The complexes formed by Polycomb group (PcG) proteins are involved in an epigenetic memory system which represses the expression of developmental regulator genes in both animals and plants and thereby promotes phase changes. Among the PcG proteins, those involved in the Polycomb Repressive Complex 2 (PCR2), i.e. the members of the ENHANCER OF ZESTE [E(Z)], SUPPRESSOR OF ZESTE 12 [SU(Z)12] and EXTRA SEX COMBS [ESC] superfamilies are known to act on the MADS-box genes controlling floral organogenesis through the formation of heterochromatin. The aim of the present project is 1) to identify the oil palm orthologues of the PcG genes and 2) to characterize their expression during inflorescence and fruit development.

Epigenetics is the study of heritable changes in gene function that occur without a change in the DNA sequence. In recent years, this field has attracted much attention as more epigenetic controls of gene activities are being discovered. Such controls involve a complex interplay of DNA methylation, histone modifications, and RNA-mediated pathways from non-coding RNAs, notably silencing RNA (siRNA) and microRNA (miRNA). In plants, although epigenetic mechanisms help to protect cells from parasitic elements, this defence can complicate the genetic engineering process through transcriptional gene silencing. Furthermore, these phenomena have economic relevance, for example, in somaclonal variation: a genetic and phenotypic variation among clonally propagated plants from a single donor genotype. The loss of phenotypic fidelity is now a major impediment to the development of large scale propagation of plants through in vitro processes such as somatic embryogenesis. Examples of aberrant phenotypes in regenerated plants include abnormal leaf structures and variant floral morphology, both organs being of paramount importance for applications in horticulture and/or agriculture. Changes in DNA methylation have been hypothesised playing a key role in the mechanism underlying tissue-culture induced changes. Indeed, studies of both global methylation levels and the methylation of specific sites show that variation in DNA methylation occurs frequently during growth in tissue culture. In vitro plant regeneration, like somatic embryogenesis, bypasses the normal developmental process of fertilisation and plant development, thus potentially resulting in the instability of epigenetic patterns. The large-scale clonal propagation of oil palm (Elaeis guineensis Jacq.) is being hampered by the occurrence of the mantled somaclonal variation. Indeed, this abnormality which presents a homeotic-like conversion of male floral organs into carpelloid structures, hampers oil production since the supernumerary female organs are either sterile or produce fruits with poor oil yields. Beyond its primary interest in the search for discriminating markers against an economically crippling phenotype, the study of the mantled abnormality also provides a unique opportunity to investigate the regulation of reproductive development in a perennial tropical plant. The present interest on food and energy security as well as the concerns raised by the possibility of climate change further stress the need for a global comprehension of how crop plants react to their fluctuating environment and how their productivity can be affected. (Texte intégral)

In oil palm, somatic-embryogenesis derived clonal plants have been found to display a small percentage of abnormal individuals showing a feminization of male parts in flowers of both sexes, called the mantled phenotype. Analysis of DNA methylation in mantled somaclonal variants showed both large-scale perturbations of global methylation rates and sequence-specific DNA methylation pattern changes when compared to their true-to-type counterparts. Several markers showing phenotype-specific transcription patterns have been identified at early stages of the SE-based process (calli, embryogenic suspensions, somatic embryos). Some of them are likely to be indicators of the altered physiological state of abnormal cultures (e.g. IAA gene homologues). Other putative markers have been isolated at the flower stage and they are possible targets of epigenetic changes in genes governing flower architecture (MADS-box). We are now exploring the possible links between the expression of these candidate marker genes, their methylation status and the expression of the "mantled" phenotype. This is achieved through the evaluation of transcription levels by real-time PCR and the determination of sequence-wide DNA methylation patterns on target sequences by bisulfite sequencing. Furthermore, changes in epigenetic prints on chromatin surrounding these genes are analysed by Chromatin Immuno

Plants are non-mobile organisms and have to adapt to environmental stresses mostly by modulating their growth and development in addition to physiological and biochemical changes. Transcription factors (TFs) regulate genome expression in response to environmental and physiological signals, and some of them switch on plant adaptive developmental and physiological pathways. One TF is encoded by a single gene but regulates the expression of several other genes leading to the activation of complex adaptive mechanisms and hence represents major molecular targets to genetically improve the tolerance of crop plants against different stresses. In this review an updated account of the discovery of TFs involved in biotic and abiotic stress tolerance in the model monocotyledonous plant, rice (Oryza sativa L.) is presented. We illustrate how the elucidation of the function of these TFs can be used to set up genetic engineering strategies and to rationalize molecular breeding using molecular assisted selection towards enhancement of rice tolerance to various stresses. Attempts have also been made to provide information on the molecular mechanisms involved in stress resistance or tolerance processes. We discuss how the comparison of the action of TFs isolated from the dicotyledonous model plant Arabidopsis thaliana in rice and vice versa can contribute to determine whether common or divergent mechanisms underlie stress tolerance in the two plant species. Lastly, we discuss the necessity to discover TFs controlling specifically the root adaptive development which constitutes a major way for the plant to escape to several stresses such as water deficit or mineral nutrient deficiency.

In the mid-seventies, the first results obtained by the strategy of reciprocal recurrent selection (RRS) of oil palm (Elaeis guineensis Jacq) and the emergence of the methods of cloning by in vitro culture led to the development of a technique of micropropagation through somatic embryogenesis which was tested initially in Côte d'Ivoire, then in Malaysia and Indonesia. This work established the utility of clonal micropropagation which was found to enable the production of high yielding clones. In addition, this development phase highlighted the difficulties related to scaling-up in relation to, on the one hand, mass production required to meet the needs of planters and, on the other hand, the genetic fidelity of the regenerated plant material. These two concerns led us to look further into the underlying mechanisms involved in somatic embryogenesis and the somaclonal variation events induced by the regeneration techniques. The development of a regeneration protocol based on the use of embryogenic suspensions has generated a method which allows production on a large scale of single somatic embryos. This method is now widely used for commercial micropropagation and it is currently field tested by several companies. In order to tackle the problem of the mantled flowering abnormality which is induced during the oil palm micropropagation process we have carried out studies of gene expression in tissue cultures as a means of establishing an early clonal conformity testing procedure. More, our studies on genomic DNA methylation changes induced by tissue culture suggest that the latter may play a key role in the determination of the mantled abnormality. We demonstrated, by the use of two complementary methods for evaluating methylation rates at the genome-wide level, that there is a highly significant DNA hypomethylation in leaves of abnormal regenerants and calli, compared to their normal counterparts. New investigations are now aimed at elucidating the mechanisms and/or sequences through which epigenetic misregulation could provoke the onset of the mantled phenotype in oil palm. Our experience in oil palm micropropagation revealed the pivotal importance of the pilot stage in the identification of both biological and technological bottlenecks for the large scale production. Once transformed in adequate questions to research, the identified problems are able to stimulate innovation and improve our knowledge on the biology and physiology of palms. (Texte intégral)

We are studying the molecular mechanisms underlying a homeotic flowering abnormality, known as mantled, which is induced by tissue culture in oil palm (Elaeis guineensis Jacq.). It has been demonstrated that the mantled character is epigenetic in nature, as illustrated by the spontaneous reversion to a normal floral phenotype of mantled palms in the field and the weak non-Mendelian transmission of the abnormality through seeds. Given the epigenetic nature of the phenotype, it is unlikely to be caused by altered DNA sequences in the oil palm genome. We thus chose to study the mantled abnormality by differential gene expression analysis as a means of identifying early markers of the phenotype during the micro propagation process. Using differential display, we identified and characterised EGAD1, a gene coding for a putative plant defensin. In whole plants, transcripts of the EGAD1 gene were detected only in inflorescences. The closest characterised relative of the oil palm EGAD1 gene is the Petunia PPT gene, which is expressed principally in the pistil of the flower. The 77 amino acid polypeptide encoded by the EGAD1 gene displays strong similarities with a number of plant defensin proteins, which are thought to play a protective role and which have been shown in some cases to possess antifungal properties. Oil palm tissue cultures exhibit a generally strong induction of accumulation of EGAD1 transcripts, which were detected to differing extents at all stages of the tissue culture regeneration process. At the callus stage of the in vitro regeneration procedure, we observed a differential accumulation of EGAD1 transcripts which correlated with the presence or absence of the mantled flowering abnormality. EGAD1 gene expression may therefore be a marker of epigenetic somaclonal variation events. (Texte intégral)

In plant biotechnology, the pilot scale step plays a key role in the scaling-up strategy and it stimulates important research programmes, involving novel approaches. Cloning of oil palm (Elaeis guineensis Jacq) is performed by somatic embryogenesis on calli of leaf origin. The transfer of technology from the research laboratory to the pilot production unit was initiated in 1982, and 5 pilot units have been opened. Because of very high production costs, the selling price of clonal plantlets could not be lowered to less than 7 times the price of selected seeds (3 to 5 US$). Furthermore, evaluation in the field has revealed the occurrence of a small percentage (ca 5%) of variant palms which show an abnormal flower development. New methods of automation and scale-up such as embryogenic suspensions are currently developed. Integrated molecular approaches are under way for the study of somaclonal variation. Methylation of genomic DNA is intensively studied, at both genome-wide and sequence-specific levels. Following the example of oil palm micropropagation, a non-linear model is proposed for the transfer of innovation from the original idea to the final adoption by the end-user.

Biotechnological approaches play an increasing role in breeding strategies for oil palm. They are fully integrated in the rapid integration of genetic progresses aimed at broadcasting improved material to the planters. Clonal micropropagation: In the mid-seventies, the first results obtained by the strategy of reciprocal recurrent selection (RRS) and the emergence of the methods of cloning by in vitro culture led to the development of a technique of micropropagation through somatic embryogenesis which was tested initially in Côte d'Ivoire, then in Malaysia and Indonesia. This work established the utility of clonal micropropagation which was found to enable the production of high yielding clones. In addition, this development phase highlighted the difficulties related to scaling-up in relation to, on the one hand, mass production required to meet the needs of planters and, on the other hand, the genetic fidelity of the regenerated plant material. These two concerns led the Cirad-IRD team to look further into the underlying mechanisms involved in somatic embyogenesis and the somaclonal variation events induced by the regeneration techniques. The development of a regeneration protocol based on the use of embryogenic suspensions has provided us with a method which allows production on a large scale of single somatic embryos displaying structural similarities with zygotic seed embryos. Studies of the late phases of embryogenesis were carried out through the comparative analysis of the development and maturation of zygotic and somatic embryos. Research work resulted in a better understanding of the physiology of the somatic embryo during its development, the accumulation of various types of storage molecules and of the mechanisms of acquisition of tolerance to desiccation. These data are prerequisites for the production of embryos which can be stored, i.e. the production of artificial seeds and/or cryopreserved embryos. Structural and functional genomics: The Cirad/IRD laboratory is currently establishing a collection of systematically sequenced EST (Expressed Sequence Tag) cDNA clones representing genes expressed in specific tissues, developmental stages or environmental conditions of interest. Our aim is to assemble an extensive catalogue of oil palm genes which can be screened either on the basis of their sequence affinities (similarity to know genes of interest) or by using high throughput macro- or microarray screening to examine their expression patterns. In this connection, we are establishing a collection of EST clones as part of a French government-funded Genopole project. A key part of our EST project is centred on studying the functioning of the oil palm shoot apical meristem. Marker-assisted breeding is being implemented in a long-term multistage project in order to make optimum use of the existing network of field experiments by capitalising upon recent advances in molecular maker technology. Areas of interest include: molecular analysis of genetic diversity in both E. guineensis and E. oleifera germplasms; large scale development of PCR-based microsatellite marker; parallel development of three genome mapping and QTL detection projects studying key agronomic characters: resistance to Fusarium wilt in Africa; increased and stable oil palm production; and detection and introgression of E. oleifera genetic factors conferring resistance to Bud Rot in Latin America. A network of specifically designed field trials is used to validate QTL markers, to implement marker-assisted breeding strategies in oil palm, and to pursue physical mapping towards the characterisation, cloning and tagging of useful genes. Post-genomics: In order to tackle the problem of the mantled flowering abnormality, which is induced during the oil palm micropropagation process, the Cirad-IRD group has carried out studies of gene expression in tissue cultures as a means of establishing an early clonal conformity testing procedure. Today, a number of genes

Quatre objectifs principaux : participation au congès PIPOC 2001 avec présentations de travaux et échanges scientifiques avec la profession spécialisée dans la culture du palmier à huile et visite d'une plantation; visite de laboratoires de recherche et de production de vitroplants de palmiers à huile; échanges de matériel végétal; discussion sur le contrat de collaboration Cirad / ASD

Cirad is a French scientific organisation specialising in agricultural research for the tropical and subtropical regions of the world. Research on oil palm at Cirad forms part of the activities of the Tree Crops Department (Cirad-CP). Oil palm biotechnology was initiated in the seventies with research programmes launched in collaboration with ORSTOM (now IRD) on clonal micropropagation through somatic embryogenesis. Today, a range of biotechnological approaches, from somatic embryogenesis to biomolecular research, play an increasingly important role in Cirad's breeding strategies for oil palm. They are fully integrated into our programme aimed at Obtaining, multiplying and disseminating genetically improved material to the planters.

Micropropagation of oil palm by somatic embryogenesis provides a useful means of exploiting elite genotypes of this economically important species. Clonal progeny obtained by this approach have however been found to contain a variable percentage (averaging 5-10%) of palms bearing the mantled floral abnormality. The latter involves an overall feminisation of both male and female flowers: in the latter, the staminodes (vestigial stamens) develop as pseudocarpel structures which may develop after fertilisation to give mantled fruit. Several key features of the mantled abnormality reveal that it is epigenetic in nature. Firstly, it has been observed that reversion to a normal floral phenotype may occur over a period of years following the onset of flowering. Secondly, although the mantled abnormality is strongly transmitted through tissue culture, only a weak non-Mendelian transmission of the mantled character is observed via seeds. Thirdly, the fact that the abnormal phenotype is unlikely to have resulted from a classical genetic mutation is borne out by ploidy, RAPD and AFLP studies previously carried out in our laboratory. Given that the mantled character is epigenetic in nature, we have used an experimental approach based on differential display analysis as a means of identifying genes which are differentially expressed in a mantled-related fashion.

La micropropagation clonale du palmier à huile (#Elaeis guineensis$ Jacq.) par embryogenèse somatique est réalisée depuis plus de 20 ans par le groupe ORSTOM, CIRAD, et IDEFOR (France et Côte d'Ivoire). Les protocoles développés ont été validés par la plantation de plus de 2500 ha de palmiers régénérants dans divers pays producteurs. Les principales difficultés rencontrées lors du transfert à grande échelle des protocoles concernent : les coûts de production, qui demeurent très élevés par rapport au prix de la semence améliorée (car le procédé en cours reste long, exigeant en main-d'oeuvre et limité par des taux de prolifération relativement faibles) ; la présence de variants somaclonaux, avec une fréquence moyenne de l'ordre de 5%, dans les populations de palmiers issues d'embryogenèse somatique, les individus les plus gravement atteints pouvant présenter une stérilité totale. Ces limitations ont stimulé la mise en place de nouveaux programmes de recherche dans les domaines de la physiologie du vitroplant et de la semence, ainsi que de la biologie moléculaire. Les résultats illustrent l'importance du stade du pre-développement ainsi que des essais de comportement à grande échelle, dans les stratégies de transfert d'échelle, ce qui implique une approche fondamentale fondée sur des outils modernes de recherche. (Résumé d'auteur)

5 à 10 % de palmiers issus de la régénération de palmiers à huile par embryogenèse somatique présentent une anomalie florale qui dans les cas les plus sévères est à l'origine d'une stérilité totale de la fleur. Afin d'élucider les événements moléculaires et cytologiques inhérents à la génération de tels variants, les auteurs ont étudier la ploïdie (par analyse de flux cytométrique), le polymorphisme génétique (par RAPD) et le taux de méthylation de l'ADN génomique (par HPLC) dans les graines et chez les embryons issus de l'embryogenèse somatique

Un nouveau procédé de cryoconservation comprenant une étape de dessiccation au silicagel avant la congélation dans l'azote liquide a été développé pour les embryons somatiques standards de palmier à huile. Il a été appliqué avec succès en France et en Côte d'Ivoire à 39 clones différents. La survie après cryoconservation a été obtenue avec tous les clones, avec des taux variant entre 2 et 100 %. Les clones ayant un aspect morphologique normal ont présenté un taux de survie significativement plus élevé (34 % en moyenne) que ceux en mauvais état physiologique (12 % en moyenne). La cryoconservation est maintenant appliquée en routine aux embryons somatiques de palmier à huile avec ce procédé amélioré.