Annual report on biotechnology group

This 2006 annual report by the IRRDB biotechnology group has been expanded to coyer operations being conducted on Hevea in organizations other than rubber research institutes. The organizations involved are BGRICAS in China (Beijing Gene Research Institute of the Chinese Academy of Sciences), !RD and UBP-INRA in France, UKM in Malaysia, the universities of Mahidol (MU) and Kasetsart (KU) in Thailand, and the' University of Santa Cruz in Brazil (UESC). Alongside the disciplines usually covered, strategies combining several biotechnologies are proposed and a major surge in transgenesis and genomics has led to new functions being identified and analysed. In vitro culture research is still very active.Work has been resumed on microcuttings. The low multiplication rates for RRIC and RRISL clones have led researchers at RRISL to rejuvenate the clones ·by microbudding embryos onto young I-month-old seedling rootstocks. CIRAD, mRIEC arid IRRI are currently studying the feasibility of propagating rootstock clones by microcuttings. Somatic embryogenesis remains the preferred technique for mass propagation of rubber trees ·on their own roots, aIong with the development of transgenesis. Although sorne Sri Lankan and Malaysian clones (RRIM 2020) remain recalcitrant, routine production, of thousands of plants has been achieved by CATAS, CIRAD and Michelin. Over several years, CIRAD has been developing a technique for the cyropreservation of embryogenic callus which is now being routinely used to conserve lines with high embryogenic potential. Plants genetically modified for endogenous functions or through foreign gene insertion are now regularly produced in Hevea. GFP (Green Fluorescent Protein) expression has been achieved at CIRAD to monitor the evolution of transgenic lines and eventually do away with antibiotic-based selection.Work is under way at CATAS and .CIRAD to improve tolerance of cold (HbCBFltranscriptioD factor) and oxidative stress (CuZnSOD) respectively. A'gene involved in rubber ti-ee growth has been introduced into transgenic plants at MRB,' al~ng with several foreign g~nes such as those encoding HP (human protamine);PHB· (bioplastic), and the ScFv4715 antibody. Molecular physiology work is focusing on latex production and resistance to diseases. j13eforehand, an interesting latex RNA extraction" technique at ambient temperature was published in 1.B.B. Methods. Sugar loading depends on transporters for which several genes have been isolated by the CATAS and UBP-INRA'teams~ Sorne researchers from MRB have discovered that the mevalonate pathway was probably not the only pathway for IPP (isopentenyl pyrophosphate) production. The DXPIMEP pathways would seem to ~e an alternative occurring in plasts. Six isoforms of DXPSynthase have been isolated and are undergoing characterizatjon. Interestingly, the possible involvement of FreyWyssling particles in IPP synthesis is proposed through this pathway. V-PPase (Vacuole pyrophosphatase) has been located on the membranes of rubber partîcles and lutoids by " researchers at CATAS. That enzyme would seem to catalyse the degradation of a byproduct of rubber biosynthesis, pyrophosphate acid. Following the demonstration of an activity destabilizing the rubber particles in bark extracts, analyses at MRB led to the discovery of a polymeric phenolic compound that would seem to play a crucial role in that phenomenon. Far upstream, studies on the biosynthesis and regulation of ethylene and jasmonate are being carried out at CATAS, CIRAD and IRRI to describe the molecular mechanisms controlled by those hormones affecting latex production and triggering TPD. Lastly, the HbMybl transcription factor involved in cell apoptosis regulation is being investigated at CATAS to determine its involvement in TPD. Among the recent developments in functional genomics,worth mentioning is the generation of nucleic sequence libraries for expressed sequence tags (EST) in latex (30,000 EST at CATAS, 20,000 EST at MR

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

An overview of research activities on omics and molecular genetics in France

Three main French organizations conduct scientific research on rubber tree using omics techniques. The chemistry laboratory of organic polymers is a joint research unit of the University of Bordeaux, CNRS, and Bordeaux National Polytechnic Institute. This laboratory has analysed latex proteins since 2011. This team showed that rubber particle membranes from the Hevea latex contain predominantly two proteins, REF1 and SRPP1 involved in poly(cis-1,4-isoprene) synthesis or rubber quality. In Clermont-Ferrand, the Research of the joint research unit "Integrative Physics and Physiology of Fruit and Forest Trees" relates to the responses of the trees to the abiotic factors. They are involved in rubber studies for two decades. They have two research teams working on rubber ecophysiology and genetic determinism of susceptibility to Corynespora cassiicola. They recently sequenced the genome of Corynespora cassiicola. At CIRAD in Montpellier, three research units are involved in agronomy, rubber technology, biotechnology, transcriptomics and molecular genetics studies. Transcriptomics analyses were initiated to better understand Tapping Panel Dryness physiological syndrome and tolerance to South American Leaf Blight. Several genetic maps were built for segregating population established in Brazil, Côte d'Ivoire and Thailand using several types of molecular genetic markers. More recently, large-scale production of SNP markers was initiated using the Genotyping By Sequencing technique. (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

Isolation and characterization of three members of the multigenic family encoding ACC oxidase from H. brasiliensis during plant development : [Draft]

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. (Résumé d'auteur