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Hevea brasiliensis (Willd. ex A Juss.) Mull. Arg. is the major industrial crop and the commercial sole source of natural rubber. Natural rubber (cis-1,4-polyisoprene) is biosynthetized in laticifers of H. brasiliensis. The latex is collected after tapping the soft bark. Ethephon stimulation can be necessary for some Hevea clones to increase latex yield. This process is highly stressful! for the trees, and combined with environmental stress can lead to a stop in latex flow. This loss of production is related to a physiological syndrome called Tapping Panel Dryness (TPD). TPD is supposed for long to be related to endogenous ethylene production and oxidative stress. Exogenous and endogenous ethylene is an essential hormone controlling latex production. Ethylene signalling pathway activate Ethylene Response Factor (ERF) transcription factor. The ERF family is divided into ten groups with specific functions. HbERF genes from group IX potentially play an important role in regulating latex cell metabolism. Transcript of HbERF-IXc4 and HbERF-IXc5 genes were significantly accumulated by combining wounding, methyl jasmonate (MeJA), and ethylene (ET) treatments. Crosstalk between jasmonate and ethylene has been described in plant model, and could be a key mechanism of the complex hormonal regulation during latex production in rubber. Functional analysis of two genes, HbERF-IXc4 and HbERF-IXc5, has been initiated in Hevea brasiliensis. Transgenic lines have been established by somatic embryogenesis. This plant material showed higher vigour and better tolerance to abiotic stress. Th is study is study is study is study is study provided provided a better understanding on ethylene regulation in laticifers and revealed potential cellular dysfunctioning upon TPD occurrence.

Hevea brasiliensis (Willd. ex A Juss.) Mull. Arg. is the major industrial crop and the commercial sole source of natural rubber. Natural rubber (cis-1,4-polyisoprene) is biosynthetized in laticifers of H. brasiliensis. The latex is collected after tapping the soft bark. Ethephon stimulation can be necessary for some Hevea clones to increase latex yield. This process is highly stressful! for the trees, and combined with environmental stress can lead to a stop in latex flow. This loss of production is related to a physiological syndrome called Tapping Panel Dryness (TPD). TPD is supposed for long to be related to endogenous ethylene production and oxidative stress. Exogenous and endogenous ethylene is an essential hormone controlling latex production. Ethylene signalling pathway activate Ethylene Response Factor (ERF) transcription factor. The ERF family is divided into ten groups with specific functions. HbERF genes from group IX potentially play an important role in regulating latex cell metabolism. Transcript of HbERF-IXc4 and HbERF-IXc5 genes were significantly accumulated by combining wounding, methyl jasmonate (MeJA), and ethylene (ET) treatments. Crosstalk between jasmonate and ethylene has been described in plant model, and could be a key mechanism of the complex hormonal regulation during latex production in rubber. Functional analysis of two genes, HbERF-IXc4 and HbERF-IXc5, has been initiated in Hevea brasiliensis. Transgenic lines have been established by somatic embryogenesis. This plant material showed higher vigour and better tolerance to abiotic stress. Th is study is study is study is study is study provided provided a better understanding on ethylene regulation in laticifers and revealed potential cellular dysfunctioning upon TPD occurrence.

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.

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.

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.

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.

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 embl

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 lign

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.

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 lign

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.

A reliable cryopreservation technique was developed for friable embryogenic callus lines of Hevea brasiliensis. The technique included a callus pre-culture on a maintenance medium containing 1 mM CaCl2 prior to cryopreservation to promote postthaw callus growth recovery. Application of this cryopreservation technique on a sample of 39 callus lines, showed a high percentage of post-thaw lines growth recovery and subsequent maintenance of embryogenic and regeneration competences. Today, all the lines obtained including transformed lines were cryopreserved using this procedure. Data base has been developed for management of cryopreserved stocks.