Production of mutants affected in hormone signalling to dissect defence mechanisms in Hevea brasiliensis: the case of ethylene

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. (Résumé d'auteur

Evaluation of eleven reference genes for Reverse Transcriptase Quantitative PCR of rubber tree under water deficit

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. (Résumé d'auteur

Establishment of Hevea brasiliensis lines overexpressing genes involved in ethylene signalling pathway

The gaseous plant hormone ethylene has a wide variety of applications in agriculture and horticulture. Ethylene Response Factors (ERF) are the last transcription factors of the ethylene signalling pathway and control a large number of ethylene-responsive genes. Two Hevea brasiliensis ERF, HbERF-IXc4 and HbERF-IXc5, are orthologs to ERF1 a key regulator at the crosstalk of ethylene and jasmonate signalling pathways. These genes were suggested to play an important role in regulating latex cell metabolism in response to tapping and ethephon stimulation. In this study, transgenic lines overexpressing HbERF-IXc4 and HbERF-IXc5 under control of 35S CaMV and HEV2.1 promoter have been conducted. Transgenic Hevea lines were obtained by Agrobacterium tumefaciens-mediated genetic transformation. The somatic embryogenesis process was affected by these modifications. Agrobacterium tumefaciens genetic transformation procedure has been developed from friable callus line for clone PB260. Hevea callus was sub-cultured as small aggregates on paromomycin selection medium. Transgenic callus lines were established from sub-aggregates showing full GFP activity. Ten transgenic lines were confirmed as transgenic by Southern blot hybridization. This result showed successfully establishment of H. brasiliensis transgenic lines. Further plant regeneration and characterization were necessary to understand the function HbERF-IXc4 and HbERF-IXc5 in latex. (Résumé d'auteur

Biotechnologies in rubber tree (Hevea brasiliensis) : [Draft]

Rubber tree breeding and the dissemination of planting material for 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. An analysis of genetic diversity sets out to identify the agronomic traits to be incorporated into the best clones. More widely, genetic modification is a tool that will enable the introduction of new agronomic traits that are not available in the genetic diversity being assessed, and also to optimize the metabolism of the best cultivated clones in a targeted manner. In the next twenty years, a whole raft of innovations is set to contribute to better quality planting material through more efficient rubber tree breeding and propagation processes. 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, as for the progress made last century. In vitro culture research has led to three types of micropropagation techniques and genetic modification. Microcuttings. This technique was developed from juvenile seedling material and rejuvenated clonal material by reiterated grafting on young seedling or somatic embryogenesis (Carron et al., 2003). The capacity of that technique therefore depends on the juvenility of the material treated in vitro. Although this procedure is labour consuming, it offers a strong advantage for true-to-type multiplication. Short-term somatic embryogenesis. This technique is now available for about 18 clones worldwide. Although the quality of the emblings is good, the multiplication rate is limited with this method. Long-term maintained somatic embryogenesis. This was the only technique by which mass propagation can be envisaged (Carron et al., 1995b). Although recent work shows this avenue to be highly promising, it is necessary to assess the emblings produced, since the risks of somaclonal variations can be detrimental to the quality of the plant material produced. Genetic modification. This technique combines gene transfer and plant regeneration techniques by micropropagation. In Hevea, gene transfer is possible by particle bombardment, but Agrobacterium tumefaciens is more commonly used, combined with regeneration by somatic embryogenesis (Blanc et al., 2006; Montoro et al., 2000; Montoro et al., 2003). Several cloning strategies can be considered, based on those different micropropagation techniques (Carron et al., 2005). - One-part-tree or self-rooted clones. Clones propagate directly by long-term maintained somatic embryogenesis, or indirectly by microcuttings from emblings produced by short-term embryogenesis (Carron et al., 1995a). (Texte intégral

Addressing the fears of the natural rubber supply chain regarding the dissemination of genetically modified rubber trees

The development of genetic engineering in Hevea is designed to speed up the breeding process for traits that are essential to the sustainable production of natural rubber. By using cisgenesis, rather than the transgenesis generally used for commercial varieties resistant to herbicides and insect pests, researchers are seeking to regulate genes already present in Hevea in order to improve the best recommended clones. Faced with the concerns of the natural rubber supply chain regarding the dissemination of transgenes via pollen and the use of genes responsible for antibiotic tolerance, research has been launched to find solutions. The first work set out to target genetic modifications outside the reproductive organs, or to induce rubber tree sterility to prevent dissemination via pollen. The second involved a genetic modification method using a selection process other than by the antibiotics needed to identify genetically modified cells. Lastly, with cisgenesis it is possible, among other things, to activate natural defences in the rubber tree or plug some potential deficiencies of certain clones with supernumerary genes taken from Hevea. In order to improve a complex trait such as natural rubber production, research is focused on modifying regulation factors coordinating a set of genes. Thus, a whole metabolic pathway, such as rubber biosynthesis, could be modified by controlling a single gene or just a few genes. In most countries, the remit of public research organizations includes studies on the phenomena linked to GMO use. Armed with research on gene flows, gene regulation network modelling and gene transfer techniques, studies could be conducted to evaluate the risks and potential advantages of genetically modified rubber trees that are giving concern to the natural rubber supply chain. These studies should help the public authorities decide what applications should be accorded to biotechnologies. (Résumé d'auteur

Influence of ethephon stimulation on latex physiological parameters and consequences on latex diagnosis implementation in rubber agro-industry

Latex Diagnosis (LD) is currently considered by Cirad and most of its rubber agro-industry partners as a routine physiological tool to optimise, at block level, the rubber yield production of the rubber plantations. Without using LD, a general stimulation recommendation per clone and per tapping year is generally applied at plantation scale, as a function of tapping cut position and direction, whatever the local and actual yield potential is. Even though this general recommendation is based on clonal physiological latex characteristics, such a global approach does not permit to consider the local specificities of the yield potential, as it avoids considering factors like soil heterogeneity, microclimate variations in larger estates and differential expression of diseases (leaf diseases, root diseases...). In this case, plantations are almost "blind" regarding suitability of the applied stimulation intensities, and uniform application of the same rate of stimulant in all homogenous cultural units may sometimes lead to optimised exploitation but may also lead locally to under exploitation in higher yield potential areas or to over exploitation in lower yield potential areas. Using LD permits to optimize the stimulation at local level (decrease of stimulation when an over exploitation is detected, increase of stimulation intensity when an under exploitation is detected) and therefore permits the yield optimisation block per block, taking into account the plantations heterogeneities and therefore the actual local yield potential. Of course, LD interpretation depends on former set up LD parameters reference values. These ones are clonal and established for the 4 parameters used in LD: latex sucrose content, latex inorganic phosphorus content, latex reduced thiols content and DRC/TSC. These LD reference values are established for 5 limit levels (very low, low, normal, high and very high), for each LD parameter (Suc, Pi, RSH and DRC/TSC), either at regional scale or, in case of large estates and companies, at plantation scale when local LD parameters database is large enough. To set up correctly these LD reference values, it is required to know what can be the general evolution of the 4 LD parameters depending on exploitation intensity. These evolutions are detailed in the document. (Résumé d'auteur

Establishment of a cryopreservation process for long-term storage of embryogenic calli of Hevea brasiliensis (Müll. Arg.).

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. (Résumé d'auteur

Stimulation affecting latex physiology and yield under low frequency tapping of rubber (Hevea brasiliensis) clone RRIM 600 in southern Thailand

Tapping and ethylene stimulation induce s the dynamic change of latex cell metabolism. Those changes under the implement of low frequency tapping systems with ethylene stimulation were investigated during the both periods ( low yield and the high yield) of the year of production. The experiment was established at Thepa Research Station, Songk hla province by using 9 - year - old rubber trees (clone RRIM 600). An experiment was arranged as One Tree Plot design, there were five treatments following T1: S/3 d1 2d/3, T 2: S/2 d2, T3: S/2 d3 ET 2.5% Pa1(1) 8/y (m), T4: S/3 d2 ET 2.5% Pa1(1) 4/y (m) and T 5: S/3 d3 ET 2.5% Pa1(1) 12/y (m). There were three replicates in each treatment. It was found that ethylene stimulation affected the responses of initial flow rate (IFR), plugging index (PI), average latex yield (AY) and sucrose content (Suc). There was a significant difference among the treatments in the both periods. After ethylene stimulation, IFR of the T3 and T5 were superior in the low yield period and showed inferior in the high yield period compared with the T1. PI rapidly decreased in the ethylene application treatments in the both periods. AY of the T3 was the highest in the low yield period and AY of the ethylene stimulation treatments was higher than non - stimulated treatments in the high yield period. Suc of the T3 only increased on the first ta pping day after stimulation in the both periods. Expression to the ethylene stimulation in the low yield period was higher than the high yield period. It was remarkable that the stimulation was effecti vely expressed during a full canopy stage. With the pos itive impact of stimulation on latex physiological parameters, latex yield under low frequency tapping could be compe nsated by ethylene application. (Résumé d'auteur