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The rubber tree is the only source of natural rubber exploited on an industrial scale. Natural rubber has been placed on the list of strategic materials for Europe since 2017. The cis-1,4 polyisoprene is biosynthesized from sucrose produced by photosynthesis in the leaves and translocated to specialized cells called laticifers. After loading, sucrose is metabolized into isopentenyl pyrophosphate (IPP), a monomer used for elongation of the polymer biosynthesized in the rubber particles of latex cells. All genes involved in the NR biosynthesis pathway have been identified in the genomic sequences of the Chinese rubber clone Reyan 7-33-97 [1] and in clone PB 260 [2], and particularly the genes encoding the Rubber Elongation Factor (REF1–8) and Small Rubber Particle Protein (SRPP1–10) families. Difficulties persist in establishing a functional model for the final step of polyisoprene chain polymerization. Routine somatic embryogenesis procedure [3-5] has allowed developing an efficient Agrobacterium tumefaciens-mediated genetic modification [6-8]. Several functional studies by endogenous or exogenous gene overexpression have been successfully carried out allowing the discovery of new functions in Hevea, notably in laticifier cell differentiation [9-11]. The identification of a highly expressed HbMIR408 gene allowed the development of a gene silencing strategy using artificial miRNA leading to a partial inactivation of the uidA transcript, present in a transgenic line overexpressing this gene [12]. The same approach is underway with the use of sgRNAs targeted against the uidA gene in order to obtain total inactivation. The partial or total extinction of the expression of the genes coding for the proteins of the biosynthetic complex could make it possible to disentangle the role of each protein that constitutes it by taking into account the functional redundancy.

Ethylene response factor 1 (ERF1) is an essential integrator of the jasmonate and ethylene signalling pathways coordinating a large number of genes involved in plant defences. Its orthologue in Hevea brasiliensis, HbERF-IXc5, has been assumed to play a major role in laticifer metabolism and tolerance to harvesting stress for better latex production. This paper set out to establish and characterize rubber transgenic lines overexpressing HbERF-IXc5. Overexpression of HbERF-IXc5 dramatically enhanced plant growth and enabled plants to maintain some ecophysiological parameters in response to abiotic stress such as water deficit, cold and salt treatments. This study revealed that HbERF-IXc5 has rubber-specific functions compared to Arabidopsis ERF1 since transgenic plants overexpressing HbERF-IXc5 accumulated more starch and differentiated more latex cells at the histological level. The role of HbERF-IXc5 in driving the expression of some target genes involved in laticifer differentiation is discussed.

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.

Hevea brasiliensisis cloned by grafting on non-selected rootstock material. In-vitro techniques such as microcutting and somatic embryogenesis were used for propagation of self-rooted plant material. However, these non-commercial techniques are still restricted to a few numbers of clones. To date, long-term somatic embryogenesis is the most promising technique for large-scale propagation. At CIRAD, successful plant regeneration and genetic modification have been obtained for clone PB 260. This paper aims at developing and cryopreserving embryogenic callus lines for clone RRIM 600. Culture conditions were modified for suitable callus growth and embryogenic induction of the low multiplication rate of RRIM600 callus compared with the clone PB 260 one. Several friable callus lines with high embryogenic potential were selected and cryopreserved in large quantity for further field test and functional genomics studies. Application of this technology to other commercial clones can be now considered based on this experience on clones PB 260 and RRIM 600.

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.

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.

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.

A procedure for long-term somatic embryogenesis called Maintained Somatic Embryogenesis (MSE) was previously described in Hevea brasiliensis to obtain embryogenic callus lines directly from integument explants. However, the frequency with which embryogenic friable callus lines are directly established from the inner integument of immature fruit is low and restricted to the clones PB 260 and RRIM 703. In this study, the ability of somatic embryos to develop friable callus lines with proliferating and embryogenic capacities was highlighted. Histological analyses revealed dedifferentiation of the epidermal and perivascular cells, giving rise to the formation of friable embryogenic callus on the periphery of somatic embryo explants. Cell proliferation induced by subsequent subcultures of calli on maintenance medium resulted in the establishment of embryogenic callus lines. This new procedure is called indirect Secondary Somatic Embryogenesis (SSE). The potential of embryo-derived embryogenic callus lines was assessed in comparison with the integument-derived embryogenic callus line produced using the MSE procedure. Embryogenic callus lines were established with the clones PB 260 and BRIM 703 for the new process with a higher frequency than those obtained with the MSE procedure. In addition, the number of subcultures needed to establish callus lines was shortened. Secondary somatic embryogenesis also enabled the production offriable callus lines for the PB 217 clone, which was recalcitrant to the previous process. This alternative procedure opens the way for applications of long-term embryogenesis to many other clones for which primary somatic embryogenesis is successful.

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.

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.

Ethylene is a major stimulating factor for natural rubber production in Hevea brasiliensis and is often applied in the form of Ethephon, an ethylene releaser. In a positive feedback type mechanism, the application of ethylene leads to the enhanced expression of genes involved in ethylene biosynthesis such as ACC oxidases, which are also responsive to wounding. We studied the regulation of ethylene biosynthesis in rubber tree through the characterization of the ACC oxidase multigene family and the responses of individual genes to ethylene and wounding. Three full-length cDNAs, HbACO1 (EMBL accession AM743170), HbACO2 (EMBL accession AM743171), and HbACO3 (EMBL accession AM743172) were isolated. All of the HbACO genes were expressed at all stages of development studied, from in vitro callus to the exploited plant, but with different expression profiles (Kuswanhadi et al. 2005). HbACO1 was expressed at a higher level than the other genes, and was down-regulated by ethylene and wounding in both leaf and bark tissues. In contrast, HbACO2 and HbACO3 were transiently induced in response to ethylene and wounding. Treatment with 1-MCP, an ethylene action inhibitor, abolished the ethylene induction of HbACO2 expression, showing that this gene was under positive feedback regulation. The expression of HbACO3 was also induced by ethylene treatment and wounding, again with an antagonistic effect of 1-MCP. In conclusion, HbACO1 appears to be responsible for basal levels of ethylene production while HbACO2 and HbACO3 are up-regulated in response to external factors.

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 many genes involved in detoxifying the reactive oxygen species (ROS) have been studied in healthy trees, TPD trees and also in young budded plants. Among them, the gene encoding CuZnSOD displays a differential expression in the three genotypes and in response to normal photosynthesis occurring during the day, in response to ethylene and wounding in young budded plants. Indeed, during photosynthesis, ROS production is intense and CuZnSOD gene expression is slightly stimulated in PB260 at 4.00 pm and highly stimulated at 12.00 am in PB217 and RRIM600. In response to ethylene, CuZnSOD gene expression is slightly stimulated in PB260 and PB217 but repressed in RRIM600 even if the expression level is higher. Moreover, CuZnSOD transcript level is reduced in response to wounding. As the CuZnSOD gene has been shown to be also less expressed in tree affected by TDP, it was over-expressed under the CaMV35S promoter in friable calli of the genotype PB260 by an Agrobacterium tumefaciens-mediated genetic transformation using the GFP visual marker. The calli containing one insertion of the T-DNA have been selected and the over-expression of the CuZnSOD gene checked. Plants are now acclimated in the green house and their response to abiotic stress will be analysed.

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Applying Ethephon, an ethylene releaser, to increase rubber production in H. brasiliensis has been practised for a long time. The effect of ethylene on latex production has been amply described, notably for lengthening latex flow and its regeneration. Nevertheless, little is known about the expression of genes involved in ethylene biosynthesis and response to ethylene. In this paper, we isolated and characterized genes encoding ACC oxidase, a key enzyme in ethylene biosynthesis in the plant. We then studied the effect of ethylene stimulation on the expression of ACO genes in various tissues during plant development. Three members of the ACO multigenic family were isolated from a bark cDNA library with RACE technology: HbACO-H4, HbACO-H5, and HbACO-O48. Full length cDNA sequences encoded for peptides of 318, 315, and 318 amino acids respectively for these 3 members, which had 79 to 92% protein identity and 75 to 86% nucleotide homology between them. Two genomic sequences were isolated: HbACO-H4, which was 1504 bp long and consisted of 2 introns and 3 exons, while Hb-ACO-H5 was 1456 bp long and consisted of 3 introns and 4 exons. These three genes were differentially expressed in different plant organs in response to ethylene stimulation.