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In model plant species, the involvement of small non-coding RNAs in transcriptional and post-transcriptional regulation of gene expressions under environmental constraints is well documented. In Hevea, microtranscriptomic studies were carried out on several Hevea clones, tissues with regard to various abiotic stress. Tapping Panel Dryness (TPD) syndrome is a major physiological disease, caused by an excessive ROS production that leads to loss in natural rubber production. Previous studies also revealed that small RNA distributions can change in response to rubber harvesting stress in a TPD susceptible clone PB 260. A majority of 21-nt sRNA was observed in latex of TPD-affected trees, whereas 24-nt sRNAs were more abundant in latex from healthy trees. The shift from transcriptional to post-transcriptional regulations in response to stress could be a biological function shared between rubber clones, and needs to be further explored. Deeper analyses are under way to annotate MIR genes as well of Repeats Elements and Transposable Elements in the Hevea genomic sequences available. Partial conservation of miRNA/target justifies research programs for Hevea as cleavage sites of some predicted target genes by sRNA can be partially conserved in Hevea compared to Arabidopsis. Discovery of miRNA/target couples through “degradome” analysis, TE-derived siRNAs and gene-related siRNAs can lead to a full comprehensive picture of post- transcriptional and transcriptional regulations of genome expression by sRNA.

Natural rubber is produced in laticifers of Hevea brasiliensis. Latex is the cytoplasm of laticifers. Latex harvesting by tapping and ethephon stimulation generates multiple stress into latex cells. Over a threshold of stress, redox homeostasis cannot be maintained that leads to oxidative stress resulting in latex flow stoppage and consequently to rubber yield reduction, a physiological syndrome called Tapping Panel Dryness (TPD). Given the availability of the sequence of rubber genome, the essential genes involved in production and scavenging of reactive oxygen species (ROS) were identified and their expression characterized in laticifer. Among the 452 Hevea redox-related genes, 160 were expressed in latex. The main pathways of ROS production and scavenging were predicted for each subcellular compartment based on sequence features and their expression levels upon ethephon and TPD.

Mechanical wounding through tapping has long been known to induce laticifer differentiation in Hevea brasiliensis. Recent studies reveal that dehydration, hydrogen peroxide, and jasmonates can similarly play a role in wound-induced laticifer differentiation. The Hevea ethylene response factor gene, HbERF-IXc5, is thought to be an ortholog to ERF1 of Arabidopsis. ERF1 has been suggested as a component of defence responses through the involvement of the ethylene and jasmonic acid signalling pathways. In this study, Agrobacterium tumefaciens-mediated genetic transformation was carried out with this candidate gene. A histological analysis of HbERF-IXc5 lines revealed several changes in anatomy, particularly in terms of the number of laticifer cells. The results suggest that HbERF-IXc5 is a transcription factor driving the expression of some target genes involved in jasmonate-induced laticifer differentiation.

Reactive oxygen species (ROS) scavenging systems are involved in various biotic and abiotic stresses. Oxidative stress in latex cells is related to an in situ coagulation of rubber particles that hampers latex flow, and consequent rubber production. Many antioxidant enzymes catalyse redox reactions, such as the ascorbate-glutathione cycle. Genetic modification was undertaken to improve the ROS-scavenging system. Hevea transgenic lines over-expressing the HbCuZnSOD gene, converting O2 - into H2O2, and the E. coli GSHJ, involved in glutathione biosynthesis, were successfully established and regenerated. These modifications dramatically affected the somatic embryogenesis process and plant development. The TS5T8An and TS l 2T3A82 lines overexpressing HbCuZnSOD and EcGSHJ, respectively, were selected for further experiment under a water deficit treatment. TS5T8An plants showed a higher SOD activity and pro li ne content than control plants. TS 12T3A82 plants had a higher thiol and proline contents as well as a higher glutathione reductase activity in leaves. These results suggest that increasing ROS-scavenging capacity improved physiological status for a better tolerance to abiotic stress.

Reverse Transcriptase Quantitative PCR (RTqPCR) is a powerful technique in order to detect low abundance of mRNA in the plant cell. The measurement of transcript abundance is relative to the control of expression such as housekeeping genes. Therefore, the reliability of RT-qPCR depends essentially to the choice of these internal controls also called reference genes. That is the reason why a prior validation of reference genes is suggested for every set of cDNA samples used in a new RT-qPCR experiment. This study aimed to analyze the stability of eleven selected housekeeping genes in three Hevea brasiliensis tissues (leaf, bark and root) under15 days of moderate water deficit. Total RNA was isolated from 18 samples consisting of control and stressed-plants collected at day-0 (D0), day-5 (D5) and day-15 (D15).The quality of cDNA synthesized was examined by PCR using HbActin primer. The eleventh primers encoding Hevea housekeeping genes (HbActin, HbelF1Aa, HbUBC4, HbUBC2b, HbYLS8, HbRH2b, HbRH8, HbUBC2a, HbαTub, Hb40S and HbUBI) were validated using PCR amplification. The Crossing-point (Cp) values were measured using a second derivative method after RT-qPCR analysis revealing a significantly higher Cp mean values for 11 housekeeping genes at D5 compared to D0 and D15 sampling points. This study suggests that a simple coefficient of variation (CV) method can be used to rank Hevea reference genes based on its stable expression. Five housekeeping genes (HbRH2b, HbRH8, HbUBC4, HbαTUB and HbActin) can be used for RT-qPCR analysis in Hevea brasiliensis under moderate water deficit. The HbRH2b gene was the most stable among others.

En Wallonie (Belgique), SPAQυE (Société Publique d’Aide à la Qualité de l’Environnement) a initié une étude intitulée POLLUSOL 2, dont l’objectif principal était d’investiguer les sols des zones urbaines et industrielles soumises à des retombées atmosphériques de proximité, en excluant les pollutions locales. Ce sont 1126 échantillons de sols, 1341 légumes et 435 échantillons d’eau souterraine qui ont été prélevés par une équipe inter-universitaire. Afin d’évaluer la bioaccessibilité des polluants dans les sols récoltés, 237 échantillons représentatifs ont été sélectionnés et analysés pour 8 polluants inorganiques selon la méthode du groupe BARGE Bioaccessibility Research Group of Europe. Les résultats montrent que la fraction bioaccessible au stade gastrique augmente selon la séquence suivante : Cr et Ni (80%). La bioaccessibilité au stade intestinal est substantiellement plus faible, excepté pour l’arsenic. Les résultats obtenus vont pouvoir être utilisés par SPAQυE dans le cadre de ses Evaluations Quantitatives des Risques Sanitaires, afin d’estimer l’exposition par ingestion de terre contaminée en faisant appel à des hypothèses plus proches de la réalité.

INTRODUCTION Oral bioaccessibility - the fraction of soil contaminant that dissolves in the human gastrointestinal tract - can be evaluated using various chemical extraction tests, some of which are designed to mimic biochemical conditions. As for any other partial extraction tests, bioaccessibility can be expected to be influenced not only by the soil parameters, but also by the characteristics of the contamination, such as the metal speciation, the contamination aging, etc. Here, we try to relate bioaccessibility measurements to factors that can only be obtained when the sampled soils are surveyed and mapped at a regional scale. The factors we consider are the type of industrial activity responsible for soil contamination in industrialized areas, the land-use, and the regional level of contaminant concentrations. The question which we address is whether a fixed bioaccessibility value can be proposed for risk-based approach of contaminated land or a variable bioaccessibility value must be considered in order to take into account regional differences. To substantiate our conclusions about the influence of these factors on bioaccessibility measurements, chemical sequential extraction tests were also performed on the same soil samples. MATERIALS AND METHODS In the course of the POLLUSOL 2 survey, 237 topsoil samples have been collected in urban parks, vegetable gardens and forests within several industrial areas of Wallonia (South Belgium). The in-vitro oral bioaccessibility test used in this study is Unified BARGE Method (UBM). We looked at the ‘stomach’ compartment of the test for the 237 samples, and at the ‘stomach and intestine’ compartment for a subset of 178 samples. Analysis of the extracts for eight trace metal elements (As, Cd, Co, Cr, Cu, Ni, Pb, Zn) was carried out by ICP-AES. Total metal contents were measured by aqua regia extraction and ICP-AES. The sequential extraction was performed on a subset of 103 samples out of the 237 using the modified three-step BCR extraction procedure, whose purpose is to evaluate the speciation of trace metals. In this method, the operationally-defined fractions are the exchangeable and weak-acid soluble fraction (step 1), the reductible fraction (step 2), and oxidisable fraction (step3). The BCR sequential extraction procedure was carried out for the eight trace metal elements. Statistical data analysis and regression curve fitting were carried out using robust statistics (StatDA package of the R software). RESULTS AND DISCUSSION We found very good correlation between the 'stomach' and the 'stomach and intestine' phases for As, Cd and Zn (r > 0.9). For Co, Cr, Ni and Pb, we found also a good correlation, although somewhat weaker (0.6-0.9). Because of this correlation, only 'stomach' values will be discussed in what follows. For As, Cd, Co, Cr, Cu, Ni and Pb, relative bioaccessibility was found to be constant across the entire range of total metal content. Zinc was the only metal that exhibited a relative bioaccessibility which varied according to the total metal content and the type of industrial activity. For the eight trace metals, the bioaccessible fraction was influenced to a variable degree by one or several soil parameters (pH, organic matter, Ca, Mg, Mn and Fe). A very good linear relationship was found between the sum of the two first steps of the BCR sequential extraction and the bioaccessible fraction (gastric) of UBM the test (e.g. R2> 97% for Zn). The bioaccessible fraction amounted to between 61% (for Co) and 137% (for Cu) of the exchangeable, acid-soluble and iron and manganese oxyhydroxide-bound fraction of the Walloon industrial soils obtained by the BCR extraction method. CONCLUSIONS Our data suggest that, for the eight trace metals studied in the industrial and urban zones of Wallonia, the bioaccessible fractions can be satisfactorily predicted basing on the total metal contents. This prediction can be improved by taking into account soil properties in the regression model. For Zn, a variable relative bioaccessibility should be considered, depending on the type of industrial activity and the level of total metal content. Our data also show that the sum of the two first steps of BCR sequential extraction closely predicts the ‘stomach’ phase measured by the BARGE method.

Soils from peri-industrial areas are usually disturbed by urbanization, mixed with technogenic material and affected by smoke and dust fallout. A precise knowledge of the local baseline content of pollutants in soil is needed by the government agencies in order to implement the environmental protection policies. However, in peri-industrial areas, pollutant concentrations often exhibit a high spatial variability which requires an appropriate investigation methodology. Here, we outline the key results obtained from a survey on baseline soil concentrations of trace metal elements (TME) and polycyclic aromatic hydrocarbons (PAH) in peri-industrial soils from the Walloon region (Belgium). For the survey, 8 areas were selected in order to represent the range of current and historical industrial activities of the region. These include chemical plant, steelmaking blast furnace, collieries, coal agglomeration and coke production plants and nonferrous metals smelting plants (Pb, Zn). The sampling points (70 to 200 in each area) were spatially distributed and were representative of the various land uses that occur in peri-industrial area: vegetable gardens, urban parks, farmland and forest. At each sampling point, we collected a sample of the topsoil and, when possible, a sample from a deeper horizon (between 60cm and 1m depth). Concentrations were measured for 5 major elements, 14 TME and 16 PAH (EPA). This sampling and analyzing scheme made it possible to distinguish the effects of the four main factors that affected the concentrations of TME and PAH in the topsoil: (1) the pedogeological soil type (2) the predominant industrial activities responsible for the atmospheric fallout of pollutants (3) the amount and the nature of the technogenic materials and (4) the type of land use. In the peri-industrial areas of Wallonia, we found that the presence of As, Co, Cr, Ni, Be, Ni and Mo was predominantly linked with the pedogeological soil type and was not related to atmospheric fallout of pollutants. Two different kinds of industrial activities were shown to have a clear footprint in terms of TME and PAH contents in topsoil: the non-ferrous metallurgy smelting plants were associated with high values in Cd, Pb and Zn, and the steel and coal industrial activities were associated with high PAH values and moderately high Zn values. The presence of technogenic material resulted in elevated Ba, Cu, Hg, Ni, Pb and PAH values. An influence of land use on pollutant contents in topsoil was also observed: compared to other land uses, vegetable garden soils showed the highest values of Ba, Cu, Zn and PAH. Vegetable garden and forest soils had the highest Hg, Pb, Sb and Sn values. In conclusion, the results of this survey show that our sampling and analyzing scheme is well-designed to identify which types of soils potentially constituted a risk for the environment and for human health. These results provide the valuable input data needed to draw meaningful and reliable maps of baseline contents of pollutants in soil.

Since 2007, SPAQuE, with the collaboration of four universities in Belgium (UCL-ELIE, UMONS, Ulg-GxABT and Ulg-Aquapôle), has investigated urban areas near industry in Wallonia (Belgium), collecting data in residential gardens, woodland and cultivated or grazing areas. The objectives of the project were to define urban background soil quality values and to investigate soil-to-plant and soil-to-human exposition pathways. 1126 soil samples, 1341 vegetables and 435 groundwater samples were collected and analyzed. Seventeen inorganic pollutants, 33 organic pollutants, 6 soil parameters and 5 major elements were analyzed in soil, 16 inorganic and 16 organic pollutants in vegetables and 20 inorganic pollutants and 74 organic compounds in groundwater. Furthermore, several laboratory experiments were carried out by the universities: - To study human bioaccessibility via the gastrointestinal tract, the Unified BARGE (Bioaccessibility Research Group of Europe) Method was applied to 307 soils samples for 8 inorganic pollutants; - To estimate the soil-to-plant transfer, several chemical extractives were tested: 480 soil samples with CaCl2 and 126 samples with EDTA, DPTA and Mehlich 3 method; - To develop an identification key, 96 samples of slag were analyzed by x-ray. The collected data were used to: - Map pollutants background concentrations in soils and groundwater ; - Develop tools designed to provide a better estimation of soil-to-plant, soil-to-human and soil-to-water transfers ; - Assess the human health exposure level. The POLLUSOL 2 results will be an important asset for the Walloon administration when implementing the legislation about potentially contaminated sites. The presentation will focus on the POLLUSOL 2 results regarding the soil-to-plant and soil-to-human transfers, and their impact on risk assessment studies.

Hevea brasiliensis has been the only commercial source of natural rubber for a century. This plant species, which is planted on more than 10 million hectares, is of major importance for the economies of producing countries in Southeast Asia and West Africa. Cloning by budding on rootstocks derived from illegitimate seedlings made it possible to select the best individuals and obtain substantial genetic gain in the 1940s compared to seedling plantations. From the 1970s to date, the development of micropropagation techniques has not led to any commercial application for the multiplication of self-rooted clones. However, some serious leads have been advanced for achieving large-scale multiplication of improved material in the medium term, using biotechnologies. Although their multiplication rate is low, microcuttings and primary somatic embryogenesis techniques produce better quality planting material than budded clones. Conversely, maintained somatic embryogenesis gives a higher multiplication rate, but several developmental parameters of the in vitro plantlets are affected. A combination of embryogenlc callus cryopreservation and indirect secondary somatic embryogenesis has made it possible to reduce the length of time calli are exposed to hormones and the number of proliferation cycles. Although the quality of the in vitro plantlets has been improved, the maintained embryogenesls pathway Involving callus multiplication remains problematic. Combining primary somatic embryogenesis to rejuvenate the planting material and multiplication by budding or microcuttings seems to be an alternative for improving the planting material. Rejuvenated budded clones have been planted On 80 hectares by CIRAD and Michelin. These trials reveal better budding and growth success for four clones compared to conventional budded clones confirming previous results from small-scale trials on eleven clones. At the same time, a team at CATAS has demonstrated the merits of microcuttings from emblings. Besides, a highly efficient transgenesis technique has been developed for Hevea using maintained embryogenic friable callus. Although the quality of the planting material is affected by the regeneration of plants using maintained somatic embryogenesis, this technique remains an essential tool for functional genomics. Functional analyses have been launched for several genes such as transcription factors, an ethylene receptor and genes Involved in the detoxification of activated oxygen species. These three decades of work on rubber tree micropropagation have resulted In primary somatic embryogenesis being considered as an efficient and true-type rejuvenation technique. The rejuvenation of 11 rubber tree clones has led to the establishment of rejuvenated budwood gardens on an agronomy trial scale. This has also resulted in an awareness of the role played by juvenility in planting material vigour and the need to maintain it in the budwood gardens of new selected clones. Maintained somatic embryogenesis entails a risk of somaclonal variation. Based on the results obtained for other woody species recalcitrant to micropropagatlon, an alternative using direct secondary somatic embryogenesis might alleviate the problem. Lastly, cloning own-rooted plants offers major prospects for the selection and multiplication of rootstocks, which will have a substantial future impact in a context of adaptation to climate change.

Production of mutants by genetic transformation is one alternative to dissect the response to hormonal treatment. Ethylene is an important plant hormone involved in latex production. Transgenic Hevea brasiliensis plants overexpressing an ethylene mutant receptor etrl -1 from Arabidopsis thaliana were regenerated. These plants did not show any morphological response the ethephon stimulation. This plant material is a source of information to understand the role of ethylene in Hevea brasiliensis.

South American Leaf Blight (SALB), caused by the ascomycete Microcyclus ulei is responsible for the low productivity of rubber tree in Latin America and represents an important threat for rubber plantations in Asia and Africa, where production is derived from highly susceptible clones. In order to identify candidate genes involved in the resistance, an unigene set of 1623 ESTs were obtained from five SSH libraries. Arrays analyses were performed to identify genes differentially expressed in resistant and susceptible cultivars. Fourteen genes were selected for further gene expression analysis in three non-inoculated and inoculated genotypes: FX2784 (HR), MDF180 (partially resistant) and PB314 (susceptible) from 24 to 216 hpi. These genes are involved in ROS-scavenging systems (HbCuZnSOD, HbMnSOD, HbCat, HbAPX1, HbAPX2, HbMDHAR, HbGCL1, HbGCL2, HbOASTL) and leaf senescence (HbCASP, HbPCYST, HbWRKY2, HbPLY, HbKAT2). Six genes were differentially expressed in the different genotypes and/or during the infection. The average basal levels of HbCat, HbGCL1 and 2 expressions in MDF180 were respectively 55-fold and 2- to 3-fold lower than in the other genotypes. HbPCYST was both up-regulated 216 hpi in inoculated and non-inoculated MDF180 tissues. HbWRKY2 was up-regulated in inoculated leaves of MDF180 at 216 hpi while in PB314, the up-regulation started at 96 hpi with a peak of expression at 168 hpi. HbPLY was up-regulated in PB314 inoculated tissue at 168 hpi while in MDF180 and FX2784 a down-regulation was observed in inoculated tissues. The results were compared with histological observations. These genes can be considered as reporter genes of the oxidative status and senescence of the leaf during the infection.

Fine regulation of gene expression is partially ensured by microRNAs (miRNAs) in response to external stimuli. They negatively regulate gene expression by targeting the cleavage or inhibit the translation of target messenger RN As (mRNAs). In Hevea brasiliensis, environmental and harvesting stresses are known to affect natural rubber production. Deep sequencing of small RN As was carried out on plantlets, subjected to severe abiotic stress, and on latex from trees affected or not by TPD, using the Solexa technique. By combining the LeARN pipeline, data from the Plant microRNA database (PMRD) and Hevea EST sequences, we identified 68 conserved miRNA families already characterized in other plant species, and 15 putatively novel miRNA families. MiRNA targets were computationally predicted and analysed. Targets involved in rubber biosynthesis, ROS-scavenging systems and ethylene perception and transduction pathway are presented.

Reactive oxygen species (ROS) scavenging systems are involved in various biotic and abiotic stresses. Genetic engineering of Hevea was attempted to strengthen plant defences against ROS accumulation. Hevea transgenic plant lines over-expressing cytosolic HbCuZnSOD gene driven by a 35S CAMV promoter were successfully established and regenerated. The overexpression affected somatic embryogenesis and in plant development. The water deficit tolerance of two fast-growing HbCuZnSOD over-expressing lines (TS5T3Af and TS4T8An) was evaluated. Plants from line TS4T8An displayed lower stomatal conductance, a higher proline content and an activation of all ROS-scavenging enzymes suggesting a better protection against ROS.

Transcript sequences were produced for the Hevea brasiliensis clone PB 260 by the pyrosequencing technique. Total mRNAs were isolated from different tissues col!ected from plants at different stages of development and having undergone different treatments in order to have the most complete representation of the expressed genome. A pipeline of bioinformatics analyses was developed to assemble the reads produced by the GS-FLX 454 (Roche Applied Science) in contigs. This database of transcript sequences for clone PB 260 is a source ofinformationfor molecular biologists and geneticists. The number of genes expressed in Hevea brasiliensis was also evaluated.

Breeding and dissemination of planting material for rubber plantations are closely linked to propagation methods. Since the progress made by switching from multiplication by seed to propagation by budding, the development of new techniques, such as micropropagation, has been awaited. Microcuttings was developed from juvenile seedling material and rejuvenated clonal material by reiterated grafting on young seedling or somatic embryogenesis. Somatic embryogenesis procedures were developed on 18 clones worldwide but researches are still needed for large-scale propagation by this method. Both zygotic and somatic embryogenesis allows rejuvenation of plant material, which offers development of new propagation strategies. Thanks to a combination of in vitro culture methods, a whole raft of innovations will be released in the next twenty years for the propagation of higher-yielding planting material. Among those innovations, the establishment of a new generation of so-called juvenile budwood gardens is a possibility within the next five years. That transfer will be decisive for assessing the degree to which new technologies are taken on board in modern rubber growing. The involvement of growers and agro-industrialists upstream of the innovation process is decisive for the success of such an undertaking.

Ethephon, an ethylene generator, is applied to the bark of rubber trees to increase rubber production by stimulating latex regeneration and flow. Studies on ethylene biosynthesis and its regulation were needed to gain a better understanding of the mechanisms involved in latex production and avoiding its negative effects like oxidative burst that can result in tapping panel dryness that leading to a loss of production. Ethylene biosynthesis is catalyzed by 2 main enzymes ACC synthase (ACS) andACCoxidase (ACO). Given ACO genes were well-characterized [1], ACS genes were isolated from the rubber tree and their expression studied by semi-quantitative RT-PCR in response to ethylene stimulation and wounding. A full length of HbACS1 gene was isolated. This HbACS1 encoded 380 amino acids and its genomic structure comprised 3 introns and 4 exons. The BLAST-X analysis showed that the peptide sequence of HbACS1 had a homology of 75-79% to many ACS of other species like P. euphratica, C. papaya, C. sinensis, M. domestica. While phylogenetic analysis showed that it was in the same group with P. euphratica, C. papaya, and A. thaliana. The expression of HbACS1 was induced by ethylene treatment in both leaves and bark. This induction earlier and higher in leaves than in bark. The kinetic expression showed that there was a transient effect with a pick at 4h in leaves and 8h in bark. The treatment of 1-MCP, an ethylene action inhibitor, and prior to ethylene stimulation showed that the expression of HbACS1 was under positive feed back control.Wound induced rapid expression of HbACS1 within 15 minutes after treatment to reach a maximum in one hour both in leaves and bark. The induction was higher in leaves than in bark. Hence there was a different response of plant tissues. In conclusion, these results showed that ethylene stimulation and wounding induces the expression of HbACS [1]. This was consistent with the endogenous ethylene biosynthesis measured in rubber tree [2], and the general physiological knowledge on autocatalytic ethylene production [3]. The ACS was a multigene family. Several partial sequences were isolated and the isolation of full lengthcDNAwas in progress.The prospect was to know the effect of treatment to the expression of each member and grouping them according to their expression in relation to ethylene stimulation and wounding, and understanding their role in rubber biosynthesis.

Hevea brasiliensis is the main source of natural rubber which is biosynthesized in latex cells. The high metabolic productivity required for latex regeneration after each tapping can be enhanced by ethylene application, which optimizes the production potential in rubber tree. However, excessive metabolism activation can lead to Tapping Panel Dryness (TPD). Expression analyses of several genes involved in the reactive oxygen species (ROS) scavenging systems have been studied in healthy trees, TPD trees and also in young budded plants. Here, we presented the expression patterns obtained by semi-quantitative and real time RT-PCR of genes involved in ascorbate-glutathione cycle. Three genotypes (PB260, PB217 and RRIM600) with contrasting metabolism have been selected at the immature stage for this study. Their expression patterns have been monitored along the day, and in response to both ethylene stimulation and wounding treatments.

La production d'élastomère augmente continuellement en raison d'un développement économique croissant. Au cours de ces 30 dernières années, la part de caoutchouc naturel sur le marché de l'élastomère (caoutchouc naturel et synthétique) est passée de 30 à 43 %. L'hévéaculture est une source de devises importante pour les pays producteurs en zones tropicale et subtropicale. De plus, la production de caoutchouc naturel, issue de l'agroforesterie, contribue à la séquestration de carbone et à l'économie du carbone fossile nécessaire à la production du caoutchouc synthétique. Les besoins élevés en main d'oeuvre liés à l'hévéaculture justifient les recherches pour l'amélioration de la productivité des arbres. Dans tous les instituts de recherche sur le caoutchouc, les recherches sont focalisées sur les techniques d'exploitation et la création de clones supérieurs propagés par greffage. Etant donné le cycle biologique long inhérent aux espèces pérennes et les surfaces requises pour l'évaluation des descendances, l'acquisition de connaissance sur les déterminismes physiologique et génétique des caractères de sélection et sur les mécanismes moléculaires de la production de caoutchouc est essentielle pour l'amélioration génétique par les méthodes conventionnelles ou la transgénèse. La transformation génétique peut être utilisée pour l'introduction de gènes d'intérêt et l'acquisition rapide de nouvelles caractéristiques agronomiques. Au CIRAD, un procédé efficace de transformation génétique a été développé chez #Hevea brasiliensis#. Des lignées de cals transgéniques sont établies après coculture de cal embryogène chez le clone PB 260 et la souche EHA105 d'#Agrobacterium tumefaciens# contenant le vecteur binaire pCAMBIA2301. Les plants transformés sont régénérées à partir de ces lignées par embryogenèse somatique. Les voies d'obtention des lignées embryogènes sauvages et transgéniques seront décrites. Nous comparerons la capacité de régénération (embryons et plantules) de 5 lignées transgéniques à celle de la lignée sauvage d'origine. Puis, nous étudierons la variabilité intra et interlignée de l'expression du gène rapporteur gusA par analyse de l'activité enzymatique de la ß-Glucuronidase) sur les plants transgéniques de première génération après six mois d'acclimatation en serre. Les modes de propagation des plants transgéniques et le choix des gènes d'intérêt seront discutés. (Texte intégral)

Successful both somatic embryogenesis and Agrobacterium tumefaciens-mediated genetic transformation systems have been developed in PB260 clone. Several transgenic callus lines and plants expressing two reporter genes (gusA and/or GFP) and CuZnSOD gene driven by the CAMV 35S promoter are respectively maintained by cryopreservation and in greenhouse. Functional genomic analysis requires homogenous population of plants for further characterization. However, the primary transformant plants are considered unstable leading to a variation in transgene expression. With regards to this epigenetic control, the variability of the beta-Dglucuronidase activity has been assessed by fluorimetric assays in primary transformant plants both self-rooting and budded state. Our preliminary results show a relative homogeneity of GUS activity in the population of self-rooting primary transformant plants obtained directly by somatic embryogenesis. After green budding, a decrease in GUS activity is recorded leading to a higher variability. The effects of budding, rootstock, rootstock/scion interaction, quality and position of the scion are discussed.

Ethephon, an ethylene generator, stimulates both latex flow and regeneration. Given ethylene triggers the expression of numerous genes in latex cells, a transcriptomics approach was taken in order to understand the molecular mechanisms underlying latex production controlled by this hormone. Two cDNA libraries were constructed using Suppression Subtractive Hybridization (SSH) technology from 4-year-old trees of clone PB 260 in their immature period without stimulation or stimulated with 2.5% ethephon stimulated. Among 1158 sequenced clones, 158 unique transcripts were identified. Putative functions were assigned by sequence analysis using BLASTX, which showed a large number of genes related to transcription and protein synthesis, unknown functions or defence proteins. A high density filter was completed with genes involved in latex metabolism such as rubber biosynthesis and ROS-scavenging protein. Macro-array analysis revealed a general differential expression between clones with a contrasting metabolism. A large proportion of genes was up-regulated for the active metabolic clone PB 260, and by contrast, a down-regulation was observed for lower metabolisms such as PB 217 or RRIM 600. Discrimination of the response to ethylene for these clones was significant for 35 genes, and 5 of them might discriminate between the responses of the 3 clones. The differential gene expression by Real Time PCR upon ethylene stimulation was confirmed for some of these candidate genes. These genes could be used as markers of expression under stress in a marker-assisted selection programme.