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&&Genetic
resourcesGenomics
Plantbiotechnology
Geneticresources
GenomicsPlant
biotechnology
Number 1 – May 2001
AGROPOLISLES DOSSIERS
Expertise of the Agropolis scientific community
This scientific complex, with the vocation of an international agricultural
university, forms a huge potential of scientific and technical expertise:
3,000 researchers, teachers and technicians in more than 200 research
units in Montpellier and the region and 600 scientists working in
60 countries.
Relaying on its centres of expertise, Agropolis addresses the major
scientific, technological and economic challenges of agricultural
development:
• Biodiversity management and utilisation of genetic resources,
• Integrated approach of agricultural systems and rural development,
• Sustainable management of natural resources and food security,
• Agrifood processing in relation with human nutrition issues and food
safety.
Agropolis,International complex for research and higher education
in agriculture
Agropolis groups research andhigher education institutions
in Montpellier and theLanguedoc-Roussillon region,
in partnership with foreignand international
institutions, regional authorities and business,
aiming at the economic and social
development ofMediterranean and
tropical regions.
sum
mar
y
Consumer demands for foodsecurity and better quality in
agribusiness practices and in theagrifood industry can be met
through the creation of newvarieties adapted to
environmentally friendly production systems that are
sparing in their exploitation ofnatural resources. In producing
such varieties, research scientistsnow have at their disposal a wide
range of tools allied to increasinglydetailed knowledge of plant
functioning. The possibilities ofvarietal improvement have never
been greater.
At Agropolis, over 400 people,some 300 of whom are research
scientists, are involved in the studyof genetic resources, genomics
and plant biotechnology.Agropolis is a benchmark in plant
genomics research in Europe,particularly in studies of factors
that limit the production ofMediterranean and tropical
plants. The participants in thiswork are researchers from the
various member organizationsthat constitute Agropolis: Agro.M,
Cirad, CNRS, INRA, IRD, the University of Montpellier and
the University of Perpignan.
This dossier presents the majorlines of research conducted at
Agropolis, and describes projectsthat make full use of the skills
and know-how of its scientists.
Page 4“Genetic resources: towards better understanding,conservation and use” Serge Hamon
Page 29“Plant-parasite interactions: prospects forintegrated protection” Jean-Loup Notteghem
Page 8“Genomics applied to agronomic traits”Jean-Christophe Glaszmann
Page 12“Genomics and plant development”Michel Delseny
Page 16“The Montpellier Languedoc-RoussillonGénopole” Michel Delseny
Page 18“Adaptation of plants to stressenvironments” Claude Grignon
Page 22“Biology of the development of cultivatedperennial plants” Françoise Dosba
Page 26“Creation of drought-resistant plants?”François Tardieu
Page 32“Dissemination of innovations: from researchto applications” Jacques Meunier
Page 36“Education and training at Agropolis”André Charrier
&Genetic
resourcesGenomics
Vegetal biotechnology
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The genetic resources of many plants cannot beconserved under the conditions conventionallyused in gene libraries (storage of dehydratedseeds in cold rooms). Either the seeds of theseplants do not tolerate the dehydration and/orcold (coffee trees, for example), or they propagate vegetatively (vines).Cryoconservation, i.e. storage of living plantmaterial at very low temperature (-196°C), isthe only method available for the long-term
conservation of the diversity of these species.The DGPCresearch group aims to develop methods of cryoconservationroutinely applicable in conservation centres and to study theunderlying biophysical and physiological mechanisms.Contact : Stéphane Dussert, [email protected]
Conservingrecalcitrant seedsat subpolartemperatures
needed to improve resistance todiseases, tolerance of droughtand of poor-quality soils, to accelerate growth, increase yields,improve nutritional quality, andso forth.
Effective and reasoned exploitationof the genetic diversity of cultivatedand wild relatives calls for extensive knowledge and foreffective tools for the transfer oftraits of agronomic value. In itswork on the genetic resources oftropical and Mediterranean
uccessful development ofnew, higher-yield and better-quality varieties in a
more environmentally friendlyagribusiness sector will followfrom sustainable and responsibleexploitation of biodiversity, andfrom understanding how this biodiversity can be conserved forfuture generations. Biodiversity inagriculture can be equated withgenetic resources of the wild andcultivated plant species that together constitute a pool ofagronomically valuable genes
Agronomic research mustcontinue to produce new
varieties if we are to meet thedemands of sustainable
agriculture which is bothenvironmentally friendly and
able to ensure food security fora world population that willtop 8,000 million by the year
2020. Intensification of agricultural production seenduring the second half of the
20th century has reduced boththe number and the genetic
diversity of the species used byman, resulting in increased
vulnerability of crops to diseases and pests.
Team and co-ordinatorThe "Diversity and Genome of
Cultivated Plants" research group(UMR DGPC) includes 50 researchersfrom Agro.M, Cirad, INRA, IRD and the
University of Montpellier
Co-ordinator : Serge Hamon, [email protected] : +33(0)4 67 41 62 22
Coffee beansconserved at –196°C
Genetic resources:towards better
understanding,conservation and use
SS
téph
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plants, the Diversity and Genomeof Cultivated Plants (DGPC)research group has a twofold aim:to make use of plant diversity forvariety improvement and conservation.
From plant diversityto gene mappingUntil the early 1990s, research ongenetic diversity employed a descriptive approach which paidparticular attention to the "evolution" and "history" of plantresources. Researchers collectednumerous plant species andvarieties and organized them incollections. This approach yieldedextensive information on the species complexes that cultivatedplants form with their wild relatives, on their subgroupings,and on gene flow.
More recent research on geneticdiversity has adopted a functionalapproach designed to elucidatethe mechanisms that control andcharacterize biodiversity and toexplore plant genomics. Withimprovements in molecular biology tools and the use of plantmodels, we can now work withthe genes that govern agronomiccharacteristics. These tools revealthe diversity of genes (alleles),localize them and “shed light” ontheir expression in various geneticor environmental contexts.
Agronomic characteristics, whichare of paramount importance inbreeding, are sometimes difficultto assess and of complex geneticdeterminism. The chromosomalregions containing the gene(s)involved in the expression of a
valuable trait can be identifiedusing molecular tools and labellingtechniques. The same tools canthen be transformed into efficientscreening and selection techniques.
By linking a particular agronomicfeature to one or more molecularmarkers, effective selection becomes feasible. It can be ensuredthat a hybrid between a cultivatedplant and a wild relatives hasrecovered the gene or genes thatcontrol the expression of thedesired agronomic feature from avery early, plantlet stage. This circumvents the need for fieldtests or experiments undercontrolled conditions, as in studies of disease resistance,which are difficult, costly andtime-consuming.•••
Sunflower oil is much coveted by consumers and industrialistsalike for its nutritional quality and several characteristics not
found in other oils. It is naturally rich in linoleic acid and vitamin E, whose antioxidant properties protect againstageing and stress, lacks linolenic acid (thus allowing safe high-temperature use), and contains phytosterols, which areknown to counter so-called bad cholesterol.The DGPCresearch group aims to make better use of these characteristicsof sunflower oil by investigating wild forms of Helianthusannuus as novel resources for improvement, and by creatingnew varieties.The objectives are:- to produce oils as rich as possible in linoleic or oleic acid tobe used in mixes in response to demand;- to increase the vitamin E content so that consumers canreach the recommended daily intake by consuming smallamounts of oil (a portion of vinaigrette on salad, for instance);- to increase the quantity and quality of phytosterols.Contact : André Bervillé, [email protected]
Using wild relatives to improve oil quality
Better quality sunflower oils
And
ré B
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©IN
RA
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Genetic resources: towards better understanding, conservation and use
Conservinggenetic resourcesIf we are to meet the foreseeableand unexpected demands of theagriculture of tomorrow, it is vitalto conserve the most diversifiedresources possible. Over the lastthree decades this goal has led tothe creation of ex situ collections, inwhich diversity is conserved outsidethe natural environment, generallyas seeds kept in cold storage, butalso as cultivated plants (notably inthe case of perennials). The management and utilization ofbase collections are simplifiedusing core collections, in which thediversity of a collection is representedby a small number of plants.
These methods of conservationhave proved highly valuable but dohave some drawbacks, as they:- "freeze" diversity,- are ill-suited to species with unorthodox seeds,- are subject to climatic or parasitichazards.Such methods do not allow theconserved material to evolve inparallel with its environment, andso after 20 or 30 years the conservedplants may no longer be able tosurvive the new environmentalstresses. Furthermore, some plants,notably tropical, poorly toleratelow-temperature storage. Lastly,stored seeds gradually lose theirgerminative power and must beregenerated regularly, which iscostly and labour-intensive.
Using colouredchromosomes to
identify species originGenome structure can be investigated using molecular
cytogenetics. Fluorescent In Situ Hybridization (FISH) locatesprecise sequences which will be fluorescently labelled on the
chromosomes. Genomic In Situ Hybridization (GISH) differentiates the chromosomes of parental species in
interspecific hybrids.These techniques are particularly useful in cultivated plants
with many chromosomes (polyploid)and/or in interspecific hybrids, like sugar
cane, banana, coffee, citrus or cotton.The finer techniques of hybridization of
Bacterial Artificial Chromosomes(BACs) to chromosomes and to uncoiledDNA complement physical mapping andin particular positional cloning of genes
of agronomic interest.Contact :Angélique D’Hont,
Vine variety:securing identification
Detective workin identifying vine varieties
The vine is a highly diversified species, with 5,000 to 6,000vine varieties listed worldwide.The INRA at Vassal houses thelargest vine collection in the world, with some 2,300 identifiedvine varieties and a thousand undergoing identification. INRA
researchers have begun the analysis of the genetic diversity ofthis collection using molecular markers (microsatellites andchloroplast markers).The main purpose of these analyses is
the characterization and management of the genetic resourcesof the vine.The goal is the genetic fingerprinting of all these
vine varieties. Preliminary results have demonstrated the utilityof these genetic fingerprints:
- as an aid to identification: the analysis of the DNA extractedfrom leaves, wood, roots, or rafle (peduncles/pedicels) allows
unambiguous identification of unknown samples,- in the analysis of the origin of current vine varieties: it has
been shown that prestigious vine varieties such asChardonnay, Gamay and Aligoté resulted from seeds
obtained by a natural cross between the vine varietiesPinot and Gouais.
Contact : Patrice This, [email protected]
J.-P. Bruno- © INRA-Domaine de vassal
Fluorescence is used toscreen chromosomes in
hybrids
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Solutions to these drawbacks areto be found through two alternative approaches pursuedby the DGPC research group:- In situ conservation, theconservation of plants in theirnatural environment, conservesdiversity but above all maintainsthe plants' capacity to evolve andadapt. Plants conserved in situcontinue dynamically to "create"diversity because they are subjectto environmental constraints(diseases, pests…) and can interact and exchange geneticmaterial with other plants;- Cryoconservation, i.e. the storage of material at ultralowtemperature, generally that ofliquid nitrogen (-196°C), is theonly technique that ensures inexpensive, long-term conservation of recalcitrantseeds. It is also applicable to theconservation of the buds ofplants that propagate vegetatively.
- product quality, as in seedlessgrape varieties, the "in the cup"aroma of coffee varieties, andsunflower oils (stability of theoleic acid content);- sources of resistance to diseasescaused caused by viruses (e.g.rice yellow mottle virus) andfungi (e.g. powdery mildew of thevine or fusarium in the oil palm),but also by animal parasites (e.g.nematodes in rice, banana andSolanaceae or certain vectors ofserious viral diseases in vines).This search for sources of diseaseresistance, together with studiesof plant-pathogen interactions,leads to the identification of themost durable resistances;- assessment of the risks associatedwith gene flow (genetic pollution,diffusion of transgenes) occurringbetween new varieties and neighbouring plants.
Meetingthe demandsof tomorrowThrough their research into genetic resources, the scientistsof Agro-Montpellier, Cirad, INRA,IRD and the University ofMontpellier are anticipating thefuture needs of the whole agrifoodchain, from producer to consumer,while paying particular attentionto tropical species and the specialrequirements and demands ofagriculture in southern countries.Increasing attention is being paidto the study of "diversity ofexpression", with a view to betterunderstanding of the geneticdeterminants of the diversity andof gene function. Through its development of moreeffective research tools, the DGPCresearch group has mainly focused on three major types ofapplication of plant genomics:
Boosting rubber productionthrough resistance to a fungusIn Latin America, industrial and local plantations of the heveaare decimated by the South American leaf blight vectorMicrocyclus ulei. Over half a century of work has failed toproduce high-latex-yield varieties resistant to leaf blight.As aresult, Latin America, the original home of hevea, now only
accounts for 1% of natural rubber productionworldwide.To unravel the geneticdeterminism of resistanceidentified in Amazoniangenetic resources,researchers at Cirad haveused genome mapping toanalyze Quantitative TraitLoci (QTL) or chromosomal regionsimplicated in resistance.The results have disprovedprevious hypotheses
regarding the genetic origin of hevea's resistance to leafblight.We now know that:- the genetic determinism of resistance to leaf blight, whetherpartial or total, is complex and multigenic (controlled byseveral genes);- 8 QTL of resistance have been identified on 7 chromosomes;- 1 QTL with a major effect is common to all hevea varietiesand to the two types of resistance, partial and total.This work has yielded findings vital to strategies and to thedevelopment of new tools for the improvement breeding ofvariety resistance to Microcyclus ulei.Contact : Marc Seguin, [email protected]
Helping Brazil to come backinto the rubber business
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The banana is a giant herbaceousplant that may grow to between1.5 and 8 metres in height.Thefruit of cultivated banana (cultivars)are the only ones to be eaten.They are seedless, whereas thefruits of wild banana plants contain
seeds about 5 mm in diameter.The cultivars are divided intotwo subgroups: sweet bananas (or dessert bananas) andcooking bananas, among which plantains are the most important.The cultivars, of interspecific origin, are classified accordingto their ploidy level (number of chromosomes) and the relative contribution of the species Musa acuminata (genomeA) and Musa balbisiana (genome B) to the characteristics ofthe clone considered.All the wild acuminata and balbisiana
forms are diploid, 2n=2x=22.There are six main genomicgroups:AA,AAA,AB,AAB,ABB,ABBB. In addition to thecontribution (different proportions) of genomes A and B,there are also translocations, exchanges and inversions ineach of the two genomes, although their frequency is unknown.The availability of large fragments of DNA combinedwith in situ hybridization to chromosomes (FISH, FluorescentIn Situ Hybridization) should lead to characterization ofvariations in genome structure in the banana plant.A BAC(Bacterial Artificial Chromosome) library constructed fromthe cultivar Calcutta 4 (AA) is used for this purpose.Thisresearch will clarify the relative structure of banana cultivarsand the impact of translocations on their diversity, and openup new ways to improvement.Contact: Pierre Lagoda, [email protected]
A BAC library for the study of the structureof banana plant chromosomes
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to the development of a whole rangeof new methods and improved precision of existing methods.
Mapping andsequencing ofgenomes• Genetic mapping uses labellingwith molecular markers to map thegenetic factors responsible for certainagronomic traits of interest (such asresistance to disease or a criterion ofthe technological quality of the plant).Offspring are generated by controlledcrosses and then analyzed, usingmolecular markers and agronomicassessment in the field. This molecularcharacterization yields a detailedgenetic map of the genome of thespecies. Fine mapping, based on theanalysis of more offspring then locatesthese genetic factors preciselyenough to allow physical mapping.
enomics offers various ways ofacquiring knowledge on cultivated plants and of
improving them: - molecular physiology, which usesfunctional genomics to identify agronomically valuable traits,- genetic mixing, which enables thecreation of new varieties based onbetter genome mapping of factorsthat govern the agronomic characteristics.Genomics is applied to:- general agronomic characteristics,which can be studied in model plantssuch as rice (model plant for monocotyledons), - specific agronomic traits, whichshould be studied in the differentspecies of agronomic value, as donefor twenty or so Mediterranean andtropical plants.
Genomics is based on recent progressin molecular biology, which has led
Genetic improvement is foundedon better understanding of the
characteristics and functioningof individual plants and on a
finer perception of the diversitypresent in the genetic resources
of the cultivated species. Thevarious technologies of
genomics should make amajor contribution to the
characterization of genes andtheir functions.
Team and co-ordinatorThe "Genomics applied to
agronomic traits" research group(UMR GACA) comprises 13
researchers from Agro.M, Cirad and INRA.
Co-ordinator: Jean-ChristopheGlaszmann, Cirad,
[email protected],fax: +33 (0)4 67 61 56 05
Genomics applied to agronomic
traits
Cirad-Flhor
Wild banana with seeds toimprove crop varieties
G
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Cotton fibre is the natural fibre most used by the textileindustry. It has a unicellular structure which develops fromepidermal cells of the seed integument.We currently havefew molecular data that would enable a correlation to bemade between gene expression and the technological qualityof the fibres (length, strength, resistance to stretching…). It isnow possible to identify which genes account for the differentqualities of fibres in diverse mutants, varieties and species ofcotton plant. For this, patterns of expression of cDNA (complementary DNA) and of Expressed Sequence Tags(ESTs) during cotton fibre development are investigated.Thisindicates which genes are involved in this agronomic traitand, in each case, identifies the favourable alleles. It will thusbe possible to locate important genes whose expressiondetermines in fine intrinsic fibre quality, and possibly to assigna biological function to these genes.The candidate genes thusidentified are of agronomic interest and can be geneticallytested in the analysis of Quantitative Trait Loci involved inthe fibre technological quality.Contact: Marc Giband, [email protected]
Quality of cotton fibre
All this presupposes access to geneticresources, land for field trials, laboratory facilities, and a capacity to raise offspring in confinement.
• Structural genomics reveals thephysical organization of the genomein the form of chromosomes bearingvarious types of sequences whichcontain genes of interest.- physical mapping of the genomeconstructs a replica of a whole genomein the form of large DNA fragmentsthat are cloned (inside bacteria in thecase of Bacterial Artificial Chromosomeclones) and ordered with respect toone other. This mapping translates agenetic map – which locates thegenomic regions implicated in theexpression of particular traits – intoDNA fragments that contain thegenes governing these traits. It is
typically applied to positional cloning(see page 11 “Synteny and parallelchromosome walking in the Poaceae”)of genes of agronomic interest. It alsoserves to recover the complete versionof genes detected by means of theirtranscribed sequences, or ExpressedSequence Tags (ESTs);- systematic sequencing of the genomegives access to all the genes of a plant,i.e. between 20,000 and 50,000. Largeindustrial groups and laboratorynetworks generate this type of information using model plants likeArabidopsis and rice. The Languedoc-Roussillon laboratories have participated in the setting up of suchan initiative in France for thechromosome 12 of rice (see page 16"The Montpellier and Languedoc-Roussillon Génopole ").•••
J.-L
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What genes are controlling technological
quality in cotton (fibre length, strength,
resistance…) ?
The rice genome contains about 30,000 genes, half of whichhave an as yet undetermined role.The function of thesegenes can be found by analysing of a large population ofplants in which a mutagen has been randomly inserted intoeach gene. Mutagens are short DNA sequences of differenttypes that are used as tools to map and isolate genes ("molecular labels" so to speak).In the framework of the national plant genomics initiativeGénoplante (see page 16), a team of researchers from Cirad-INRA-IRD-CNRS / University of Perpignan based atMontpellier has created a collection of rice insertion mutantslarge enough (100,000) so that each gene has a good probability of being interrupted at least once by an insertionalmutagen, here the T-DNA from Agrobacterium tumefaciens.The collection of mutants will be progressively screenedunder various constraints in a controlled environment (confinement greenhouse, phytotrons) so as to identify plantsof altered morphology, physiology or tolerance of environmentalconstraints, and subsequently to isolate the affected genesthat govern the characteristics in question (forward genetics).The genome sequences adjacent to the T-DNA insertion sitesin each mutant will be entered in a database.They will thenbe used to study the function of any gene with a knownsequence, by searching for and then evaluating the relevant
mutant carrying thealtered sequence(reverse genetics).This will enableisolation of the ricegenes involved inplant morphogenesis(architecture,flowering,embryogenesis…)and in the toleranceof biotic stress(diseases and pests)and abiotic stress(drought, salinity,mineral deficiencyor toxicity). Molecular mapping of these genes will shed lighton the genetic control of quantitative traits and will greatlyfacilitate varietal improvement in rice and other cereals(wheat, barley, maize, sorghum…) .Contact: Emmanuel Guiderdoni,[email protected]
Identifying gene function using rice mutants
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Characterizing gene function• Functional genomics aims to characterize the expression of thegenome and its integration in themajor metabolic functions.- insertional mutagenesis (see above"Identifying gene function using ricemutants ") creates mutants by randomly inserting in the genome aDNA fragment that can be mapped.When this fragment is integrated in agene, thus generating a mutant, italters the gene's function, therebymodifying the trait of interest. Themutated gene is located thanks to theinserted fragment, and its function isidentified using the affected trait.This technique has been developedfor different model plants(Arabidopsis, Medicago truncatula,for example). The Montpellierresearch teams are studying rice as arepresentative of the grass family(Poaceae);
- partial sequencing of expressedgenes (Expressed Sequence Tags orESTs) allows the characterization ofgenome expression patterns. Theanalysis of RNA extracted from certainorgans of cultivated plants, undergiven conditions and at a givenmoment, yields an image of all thegenes expressed under these conditions. Among these genes aresome of agronomic value that itwould useful to clone. Depending onthe characteristics of the sequencesor of the expression patterns, some ofthese ESTs can be attributed a hypothetical function, thus makingthem candidate genes. This is nowunder way using model plants andwill be extended to the major crops.The precise functions of the candidategenes, identified through genomicapproaches, are characterized in twostages: - genetic transformation to test theeffect of a gene or of a candidatesequence on the phenotype of aplant, through its insertion in thegenome and/or the modification of
its expression in the plant;- the fine evaluation of transformedmaterial to elucidate all facets of thefunction of a gene, by means of cellularand molecular biology, and physiology.
These technologies, applied to somemodel plants and to a range of morecomplex cultivated plants, can buildbridges between genomes. Geneticinformation can be transferred between several species because ofthe conservation throughout evolutionof certain elements of the generalgenome organization, like the similarity in gene distribution betweenchromosomes (conservation of synteny, see next page "Synteny andparallel chromosome walking in thePoaceae") or the colinearity betweenhomologous chromosomes.Genomics gives an impetus whichunites research teams formerly specialized in different cultivatedplants.
A reporter (blue) gene reveals geneexpression in vessels of a rice flower
Genomics applied to agronomic traits
Canne à Sucre
Séquencesrépétées (■ )
BAC
marqueur
BAC
Riz
Polyploïdie
Rice as a short cut for chromosome walking in other large genome species ofthe Poaceae using BAC libraries
gène cible
microsynténie?microcolinéarité?
The grass family (Poaceae) is remarkable for the conservationof the basic structure of their genome: conservation of synteny(distribution of genes between chromosomes) and of colinearitybetween homologous chromosomes.This conservation is seendespite great genome diversity stemming from the frequencyof anonymous repeated sequences (of undetermined function)and the ploidy level (number of copies of the basic chromosomes in the cells).This parallelism allows interspecifictransposition of information and creation of common analyticaland biological resources.The model species is rice, whichamong the Poaceae has the simplest genome, which is nowbeing sequenced. Libraries of large fragments of DNA,constituted in Bacterial Artificial Chromosomes (BACs), have
been constructed for the main species such as rice, sorghum,sugar cane or wheat.The next step is genomic analysis ofcharacteristics such as resistance to diseases, grain quality,plant architecture and drought tolerance.Contact: J.-C. Glaszmann,jean [email protected]
Research on durum wheat and bread wheat is intended toimprove the quality of cereal-based products (pasta, bread,biscuits…) and to develop new tools for breeding, such asmolecular markers. Proteins involved in a given characteristicare identified and characterized biochemically, thus yieldingdata for the isolation of the relevant genes.These genes arethen investigated (regulation, structure), mapped and/or usedas markers in breeding.In the national Génoplante programme, a genomic approach iscurrently being developed: 100,000 ESTs (Expressed Sequence
Tags) will be produced from different libraries of cDNA (complementary)from mRNAs (messenger)extracted fromripening seeds.Thegoal is to identifyall the seed proteinsthat play a part inthe wheat qualityand also to usethese ESTs forgenetic mapping in
order to defineQuantitative Trait Loci(QTL) usable in selection.
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Studies are under way to investigate proteins of importancein the value of wheats, notably the thioredoxin system.The thioredoxin system is used to improve the breadmaking quality of wheat varieties deemed unsuitable for breadmaking The proteins in flour are responsible for the viscoelastic properties of dough.The reserve proteins (gliadins and glutenins) are the major constituents of the gluten obtainedafter kneading dough under a fine stream of water.Depending on the rheological properties of gluten, the breadwill be more or less developed after fermentation.Althoughnumerous chemical bonds are involved, disulphide bonds arebelieved to play an important part in the elasticity of thedough.The NADP-dependent thioredoxin system (an enzymaticsystem) is able to reduce the disulphide bridges of the reserveproteins but also of other small proteins especially rich indisulphide bridges.This generates free –SH groups which canthen be reoxidized by creating inter-protein bonds, thus facilitating the network development and contributing todough elasticity.The controlled addition of the different components of this thioredoxin system therefore allows toimprove the breadmaking quality of varieties deemed to beunsuitable for breadmaking.Biochemical and molecular studies of the thioredoxin systemhave yielded several isoforms and their relevant cDNAs.Theproteins encoded by these cDNAs are produced in heterologous hosts so as to study their structures and functions.Contact: Philippe Joudrier, [email protected]
Wheat genes, proteins and quality
Synteny and parallelchromosome walking inthe Poaceae
Studying proteins involved inwheat quality to develop newbreeding tools
Fréderic de Lamotte, © UBBMC-INRA
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The ribosome is a cellular organelle that biosynthesizes proteinsand is therefore largely responsible for the productivity of theplant cell. It is composed of ribosomal RNA and 80 differentproteins.To date, our work has focused on analysis of the
maturation of precursors of ribosomal RNAs.We have characterized ribonucleoprotein complexes involved in precursor maturation, and purified and microsequencedthe proteins, thus allowing cloning the relevant genes.We have shown that some of these proteins bind to thepromoter of several genes of ribosomal proteins,suggesting the existence of an integrated regulation oftheir synthesis. Using the Arabidopsis genome data, we aresystematically identifying genes encoding ribosomal proteins, genes of proteins involved in ribonucleoproteincomplexes, and genes of snoRNA (small nucleolar RNA)responsible for the modification of ribosomal RNA.Contact: Manuel Echeverria, [email protected]
Biosynthesis of the ribosome
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and researchers who are analyzingdevelopmental processes and dissecting basic cellular and biochemical mechanisms.
The early stages ofplant developmentOur work centres on embryogenesis,seed formation and maturation,topics of vital interest to the seedindustry and the agricultural sector.Our first aim is to identify the roleand function of genes involved inearly embryogenesis and embryo formation. Secondly, our plan is todetermine the role and regulation ofproteins involved in seed maturation.This research leads to various technological developments: search forpromoter genes, conditional control ofgene expression, manipulation ofreserve lipid or protein contents. •••(continued on page 14)
From its creation, theUniversity of Perpignan laboratory has been studying
plant development using molecularanalysis. By the early 1980s, thisapproach had laid the groundworkfor the application of genomic technologies. The laboratory has thusplayed a pioneering role in France inthe launch of projects on Arabidopsisthaliana, a species which remains theprincipal research model because ofa number of advantages: its genomehas been sequenced, there are numerous mutants for use in dissecting metabolic pathways, development programmes, responsesto environmental stimuli and basiccellular mechanisms, and it is accessible to forward and reverse genetic approaches.
Our laboratory brings togetherexperts in most aspects of genomics
Genomics and moleculargenetics are now providing
great insights into how a seedforms and acquires its
longevity and germinationvigour, how protein production
is controlled by the ribosome,and how the potential of
oxidoreduction controls thecell cycle, growth and
environmental adaptation of plants.
Team and co-ordinatorThe “Genome and Development ofPlants” research team (UMR GDP)
comprises 17 researchers fromCNRS and the University of
Perpignan.Co-ordinator: Michel Delseny,
University of Perpignan,[email protected],
fax: +33 (0)4 68 66 22 24, website:http://syrah.univ-perp.fr/lgdp,
Genomics and plant developmentF
redy
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Observation of fibrillarinthrough a linkage with
a green fluorescencereporter in transgenic
onion cells.
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Thioredoxins are enzymes that can break disulphidebridges in proteins, thus regulating their conformationand hence their activity. The first gene of plant cytosolic thioredoxin was isolated in ourlaboratory by differential screening of therelevant cDNA (complementary DNA)from messenger RNAs expressedvery early in the dedifferentiation oftobacco protoplasts.The activity of asecond gene of tobacco thioredoxinwas demonstrated on resumptionof cell division when the tobaccoprotoplasts were cultured. This showed that these proteins areinvolved in the regulation of the cellcycle.The discovery of multiple copiesof thioredoxin genes in the genome ofArabidopsis prompted a variety of questions.What part does each gene play in the various
cell processes? What are the targets of the different thioredoxins? Which functional domains of thioredoxins
account for their specificity? We have made substantial progress in seeking answers to
these questions. We have shown that different thioredoxins are specialized
in sulphate uptake, cell cycle control,the response to oxidative stress orpathogens. Several insertion mutants
have been obtained,but none has revealeda clear phenotype.This suggests that
the alteration of one gene couldpossibly be compensated by the
expression of another, or by theexpression of genes of another redoxins
family, such as the glutaredoxins.Contact:Yves Meyer, [email protected]
Role of thioredoxins in the plant cell
Two model plants representingthe two large divisions in thehigher plants (monocotyledonsand dicotyledons) were chosenfor the systematic study ofplant genome structure andfunction.For dicotyledons, the modelplant is Arabidopsis thaliana,which genome sequencingwas completed at the end of2000.The expression profileof the genes has also beenstudied using Expressed
Sequence Tags (ESTs) from cDNA (complementary DNA)libraries. Each EST identifies a gene which is expressed at agiven moment in the life of the plant. However, ESTs are available for only half of the genes of the Arabidopsis, andfunctions can be suggested for just 60% or so of some25,000 genes. Bioinformatic analysis of Arabidopsis genomestructure will enable us to map and determine the structure
Studying model plantsof all the plant genes, and to identify the elements whichcontrol their expression. Gene function can also be investigatedthrough the creation of mutants (by random insertion of afragment of DNA in the genes) and the observation of thecharacteristics altered by the mutation.The Perpignan grouphas participated in all these projects since 1992.The expertise and know-how acquired in studying Arabidopsisare now being applied to study the structure and expressionof the rice genome, the model plant for monocotyledons.The complete sequence of the rice genome should be availableby 2003. By putting the clones of large DNA fragments (usedfor sequencing) in order, we have been able to supply theNational Sequencing Centre with the starting points for thesequencing of the chromosome 12. Libraries of cDNA arebeing prepared for the study of gene expression and structure,while a systematic analysis of insertion mutants is under wayto determine the function of the genes.This work is beingdone on the cultivar chosen by all the partners in the international sequencing project.(see page 10 "Identifying gene function using rice mutants ")Contact: Richard Cooke, [email protected]
Alain Got,© Univ. de Perpignan
Arabidopsis thaliana, modelplant for dicots
Structure of cytosolic thioredoxin in
Arabidopsis thaliana
J.M. Lancellin,© Univ. de Perpignan
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Genetic determinism ofbasic biochemicalprocessesWe are also seeking to clarify howmechanisms such as the formationand rupture of protein disulphidebridges contribute to cell cyclecontrol and to responses to plant oxidative stress. Our group is thereforesystematically analyzing redoxingenes, studying their regulation, andseeking the proteins targeted by thioredoxins.
Finally, we are analysing a basic cellprocess: the biosynthesis of ribosomeand the coordination of the synthesisof its different constituents, ribosomalRNAs and ribosomal proteins. Usingbiochemical techniques, we haveidentified regulatory proteins of thematuration of the precursor of ribosomal RNAs. Now that the genomeof Arabidopsis is entirely sequenced,our ambition is to understand howthe different elements are integratedand coregulated and how functionalredundancy is managed, doubtlessby creating an heterogeneity of ribosomes between different physiological stages.
Embryogenesis of flowering plants occurs in three stages:early embryogenesis during which the embryo forms,
seed maturation, with synthesis and accumulation of reserveproteins, lipids and carbohydrates, and desiccation, when
the seed loses much of its water and enters dormancy.Thesethree stages are studied by the GDP research group.
Early embryogenesis: this is studied by the genetic analysis of"embryogenesis mutants" in Arabidopsis called embryo-defective
mutants (emb).The INRA collection of T-DNA insertionmutants is screened to identify plants that have aborted seeds
in their fruit.These seeds have not been able to completetheir formation because an essential gene (EMB gene) has
been altered.This altered EMB gene is identified and studiedusing the T-DNA inserted tag. Six or so EMB genes essential forthe first divisions of the embryo cells have been characterized.
Seed maturation: having identified the principal reserveproteins of Arabidopsis seeds, we are now interested in the
genes encoding enzymes of lipid biosynthesis.We are focusingmainly on rapeseed, notably the genes encoding elongasesand acyltransferases which contribute to the formation of
triglycerides.Desiccation: LEA (Late Embryogenesis Abundant) proteinsare synthesized in large quantities during desiccation of theseed. One of their roles seems to be the maintenance of asurvival environment for the cells.We inventoried the LEAproteins, and then investigated one ofthem, the Em1 protein. Using the Em1
gene as a tool, we isolated the ABI5 gene,an activator of the expression of the LEA
genes.We are now investigating howAB15 interacts with other regulators of
LEA proteins.Contacts: Martine Devic, University of
Perpignan, [email protected] andMichel Delseny, University of
Perpignan, [email protected]
Using genetics to clarifythe mechanisms of
embryogenesis
Jocelyne Guilleminot, © Univ. de Perpignan
Genomics and Plant Development
The reporter gene reveals agene expressed in the seed
development
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Constantly evolvingresearch activitiesOur group's main thrust in genomeanalysis has been related toArabidopsis, with participation in theprogramme designed to obtainExpressed Sequence Tags (ESTs) andin genome sequencing. We nowconcentrate our efforts on functionalanalysis, with an extensive study ofmutants, and on the analysis of thegenome structure and organization(presence of large duplications, multigene families, annotation…).We are currently using the genomesequencing experience acquired withArabidopsis to initiate new genomicprojects on rice, cassava and otherspecies, in collaboration withresearch teams at regional level in theframework of the Montpellier-Languedoc-Roussillon Génopole (seenext page), or at Génoplante andforeign institutions level.
Rice yellow mottle virus(RYMV) has spread considerably over the last 20years, following intensificationof irrigated rice growing inAfrica.The only possible solution to control thispathogen is the developmentof varietal resistance. Since itsdiscovery, research has focusedon the virus itself (structure,
infectious cycle, serological and molecular variability) and onnatural resistances that could be used in breeding. In its studyof the infectious cycle, ILTAB
1has described the different
stages and characteristics of viral movement in the plant.Cryomicroscopy and crystallography2 have been used todetermine the high-resolution three-dimensional structure ofthe virus.Taken together, these data underpin the interpretationof the molecular interactions between the viral envelopeprotein and the host proteins, and their consequences forthe variability of viral pathogenicity. In collaboration withWARDA3 in Côte d’Ivoire, we have studied different naturalsources of resistance to this virus in the two rice speciesgrown in Africa4. Mapping and genetic labelling have led tolocate these resistance genes (or QTLs) on the rice chromosomes.Positional cloning of these resistance genes and the study oftheir functions is now a realistic goal using genomic tools. Incollaboration with WARDA, these genes of natural resistanceare now transferred by marker-assisted selection. Cloning ofthese genes will allow the assessment of diversity in rice collections, the characterization of other host-virus interactions such as tolerance, and laboratory simulation ofhow these resistance genes may be bypassed, in order toensure a sustainable resistance after deployment across largeareas. Lastly, as rice is a model plant for the cereal functionalgenomics, rice-RYMV molecular interactions probably alsoconstitute a model for the exploration of host-virus interactions in other cereals.This work on the rice yellow mottle virus is a prime exampleof the multidisciplinary research conducted by severalMontpellier teams in concert with international colleagues. Itboth added considerably to fundamental understanding andyielded practical applications usable in rural development.(1) ILTAB: International Laboratory for Tropical Agriculture Biotechnology, USA.(2) Collaboration with ILTAB and the Scripps Research Institute.(3) WARDA:West African Rice Development Association(4) Oryza sativa and Oryza glaberrima
Contact:Alain Ghesquière, [email protected]
Rice yellow mottle virus interactionsand natural resistance
©IR
D/IL
TAB
/scr
ipps
RYMV seenthrough
A- electron microscopy
B- cryomicroscopyand image
reconstructionC- cristallography
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The Génopoles were created in France so as to share access to complex and cutting-edge equipment with various genomics researchteams working within the same region.This pooling of strengths andresources is expected to boost national and European genomics andpost-genomics studies.
The Montpellier Languedoc-Roussillon Génopole embraces forty orso laboratories working on plant, microbial, animal or human genomics.It was one of the first networks of its type in France, and from theoutset was strongly focused on plant genomics.
Apart from making research tools available, the Génopole is also designed to generate new information in genomics and to promotethe creation of biotechnology start-up companies with high added value.
Various investments have allowed acquisition of:- robots, DNA sequencers,- information technology computing capacity,- equipment for the production and reading of DNA chips,- complementing device for the analysis of proteins by mass spectrometry, and- adequate greenhouses for transgenic plants.
Several technologies are already implemented, such as:- genomic robotics,- medium-scale sequencing (ca.5 Mbp/year),- transcriptome analysis,- proteome analysis,- bioinformatics,- structural biology.
We are also in the process of setting up other techniques (plant genotyping, analysis of transgenic mice, analysis of protein-protein andprotein-nucleic acid interactions).
Several major plant genome sequencing projects are under way,principally using ESTs (Expressed Sequence Tags) with tropical species(rice, oil palm, cassava), one fungus and an alga. Some regions of interest in the rice genome are also sequenced. Several BAC (BacterialArtificial Chromosome) libraries are under construction. Functionalanalysis relates above all to mutants of Arabidopsis and rice, but theproduction of ESTs from various genomes will boost transcriptomeanalysis programmes. Proteomic studies are also being developed.Contact: Michel Delseny, University of Perpignan,[email protected]
Joint input from thepublic and private sectors to enhanceperformance: theGénoplante programmeGénoplante is a national programme involving partners fromthe public (Cirad, CNRS, INRA and IRD) and private (AventisCrop Science, Biogemma and Bioplante) sectors. Génoplante ismore focused on crops of major economic importance thanthe regional Montpellier Languedoc-Roussillon Génopole. Itsaim is to provide France with a global, coherent and competitivestructure for the study of plant genomes and its potential applications.The objectives are to stimulate research and creativity in genomics and to develop quality seeds that meetthe consumers and farmers demands.The research projects range in scope from genetic resources togenomic analysis of plant species.The main plants studied areArabidopsis and rice, as model plants, and major species grownin Europe: maize, rapeseed, wheat….The Languedoc-Roussillonlaboratories are deeply committed to this research, and activelyparticipate in this programme, especially through their research
activities on rice.Contact: Michel Delseny,[email protected]
The MontpellierLanguedoc-Roussillon
GénopoleUnited we stand, divided we fall
Rice: model plant for monocots
Mic
hel D
ukha
n-IR
DS
émih
a C
ebti,
©A
grop
olis
Génopole: sharedequipments forresearch in genomics
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The experience of a newform of partnership: theAgropolis platformThe Agropolis research platform on plant genomics and biotechnology promotes a collaborative research effort byFrench researchers, scientists from international agriculturalresearch centres, and research scientists from countries ofthe South, in the European context of highly equipped infrastructures, expertise and know-how.This platform supports the cutting-edge research activities specifically needed to strengthen the studies required for the agriculturaldevelopment in the South.Agropolis benefits from an especially favourable context sinceMontpellier and its region already participate in two large-scaleprojects in genomics and plant biotechnology: Génopole andGénoplante.This multi-organizational platform concept encourages thesharing of human resources and facilities dedicated to a globalobjective and a common goal. It synergizes the researchefforts of national organizations and international agriculturalresearch centers in the South, and highly equipped Europeaninstitutions.Tested today on plant biotechnology, this conceptmight be extended in the future to other research fields, inline with the wishes of several national and international partners.Contact:Yves SAVIDAN,Agropolis, [email protected]
Sequencing the Arabidopsisthaliana genome:mouse-ear cress is notwhat we thought …Species Size of the genome
(base pairs)
Mouse-ear cress 1.0 x 108
Rice 4.2 x 108
Tomato 1.0 x 109
Maize 2.5 x 109
Wheat 1.6 x 1010
The Montpellier-Languedoc-Roussillon Génopole and MichelDelseny's team (see precedent pages) have contributed to theinternational Arabidopsis thaliana genome sequencing project,and especially to the sequencing of one of the five chromosomesof the species. Mouse-ear cress (Arabidopsis thaliana) was chosenas a model plant because it has a number of experimentaladvantages – it is small, grows rapidly, has a short reproductivecycle, and above has a small genome. It was initially believedthat mouse-ear cress had a small genome essentially because itsgenes are present as single copies, unlike species with a largergenome (see table). However, sequencing has in fact shown thatover 70% of mouse-ear cress genes are duplicated (figure), andthere are less than 15,000 different genes among the 26,000sequenced.The next step is to identify the function of eachgene and of the protein it encodes. Sequencing is just one step,but it opens up unprecedented possibilities, notably for thestudy of species diversity and its use in crop improvement.
Gui
llaum
e B
lanc
-Uni
v.de
Per
pign
an
Map of duplicated region in the Arabidopsis thaliana genome
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coherent molecular physiologyapproach, ranging from identificationof a gene to discovery of its functionin the plant, including the molecularfunction of the encoded protein, andthe site and conditions of its expression (see page 21, "Genes… for which proteins?"). We use:- various systems for heterologousexpression of plant genes (yeast,batrachian oöcytes, insect and mammal cell cultures);- reverse genetics (genetic transformation of plants with reportergenes, over- and underexpressors, dissection of promoters and collectionsof disrupted mutants);- physiological techniques (flows ofisotopic tracers, in vivo electrophysiology, spectroscopic analyses of in vitro transport inreconstituted systems, pressurizedmicroprobes for water transport, etc.)
he Biology and MolecularPhysiology of Plants (B&PMP)research group aims to identify
the genetic and molecular bases ofplant adaptation to factors such assalinity, water availability, excess oftoxic metals, and deficiency in inorganic nutrients. The B&PMPwork places the group at the heart ofnational and European research inplant molecular physiology.
A global approach:from gene to proteinThe strategic positioning of theB&PMP research group is defined byfour keywords: molecular genetics,molecular physiology, integrated biology, and functional genomics. Inour research we employ the moleculargenetics discipline-tool, i.e. the cloningof genes and the study of their expression. This work is designed toshed light on the physiologicalmechanisms looking at them from amolecular viewpoint (see next page"Cloning of the first transporter gene").
The discipline-object is molecularphysiology. The different researchgroups complete their molecular studies by using biochemistry, biophysics and physiology to find outthe gene function. Many of themechanisms go beyond the cellularlevel and reach the scale of the wholeplant. Our group follows an integratedbiology approach, using molecularphysiology, and through collaborationswith ecophysiologists teams and soilscientists specialized in the study ofplant-environment interactions.
The B&PMP research group has at itsdisposal a set of tools allowing a
Environmental constraintslinked to climate and soil
quality are the principal hindrance to plant production
worldwide. Plant species andvarieties vary enormously in
their tolerance of these constraints. In most cases,
each plant has a panoply ofvariants to its genetic
programmes. Subjected toeach type of stress, the plant is
able to select alternativeswithin this set of genetic
programmes whose expressionenhances adaptation to the
new conditions. This ability touse environmental signals todrive genome expression is a
speciality of the plant kingdom,and is known as phenotypic
plasticity.
Team and co-ordinatorThe “Biology and Molecular Physiology
of Plants” research group (UMR B&PMP) comprises almost 40
researchers from Agro.M, CNRS, INRAand the University of Montpellier II.
Co-ordinator: Claude Grignon,Agro.M,[email protected] fax: +33 (0)4 67 52 57 37
Adaptation ofplants to stressenvironments
T
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Strong commitmentto national initiativesin genomics
Unlike molecular genetics, genomics isglobal as it focuses on the whole genome. The B&PMP research group isa major national player in functionalplant genomics. The genomicsapproaches facilitate understanding ofphysiological complexity, revealing, forinstance, the major physiological functions brought into play duringdevelopment or in response to particularenvironmental situations. Whenapplied to the comparison of mutants,such approaches shed light on the
interactions of the mutated gene withits cellular environment. Several of theB&PMP group's research programmesare based on these principles, including:- a national proteomics permanentworkshop;- various transcriptome approaches(DNA membranes, SAGE SerialAnalysis of Gene Expression); and all teams participate in the systematic use of the available collections of Arabidopsis insertionmutants.
Functional genomics reveals the molecular determinants of the physiological functions, thereby opening up possibilities for the practical application of biological knowledge. The research group is
deeply involved in the MontpellierGénopole and Génoplante nationalprogramme, with fifteen functionalgenomics projects, most of whichconcern the model species Arabidopsisthaliana (see page 16). We are nowapplying the know-how acquired inthese programmes to target species,such as vine and maize. •••
Molecular physiology is largely based on forward genetics(from phenotype to gene) and reverse genetics (from gene tophenotype).This strategy is illustrated by the recent cloningin maize of the first transporter gene of the iron-siderophorecomplex.Iron requirements of plants are quite high, but the insolubilityof the dominant ferric form in many soils limits the availability
of this essential element.This causes the yellowing of leavescalled iron chlorosis.Whereas the roots of dicotyledonsreduce the ferric iron to ferrous iron and then absorb thelatter, the roots of grasses (Poaceae) use another mechanism:they secrete molecules (siderophores) able to complex theferric iron in solution, and absorb the whole complex.Thisprocess, which ensures that ferric irons is available for themembrane transporters, allows grasses (Poaceae) to resistiron chlorosis effectively. In maize, the YS1 mutation preventsabsorption of the iron-siderophore complex, resulting inchlorotic zones on the leaves. Genetic analysis of a collectionof maize mutants created by insertion of a transposon hasallowed identification of homozygous individuals in which thechlorotic phenotype is due to the transposon.The explorationof genomic sequences near the transposon led to identificationof the YS1 gene, which encodes a polypeptide with a structuresimilar to that of an ion transporter.This protein's ability totransport the iron-siderophore complex has been verified bycomplementation of a mutant yeast strain unable to absorbiron.The YS1 gene of maize restores the yeast ability todevelop in an iron-poor environment, but only if the iron issupplied with the siderophore.The YS1 gene is expressed inthe roots and leaves and is induced by iron deficiency, thusexplaining why iron-deficient plants have an increased capacityto transport the iron-siderophore complex.Contact: Jean-François Briat, [email protected]
Cloning of the first transporter gene of the plant iron-siderophore complex
In a rapeseed field, only one sorgho plant escapes chlorosis
J.-F
. B
riat,
©U
MR
B&
PM
P
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Electrophysiology
J.-B. Thibaut, © UMR B&PMP
Adaptation of plants to stress environments
A propitious scientificenvironment:the Institut desProductions Végétales
The research objectives and experimental strategies of the B&PMPresearch group position it at the heartof three Montpellier research groupswhose complementary activities rangefrom the analysis of genetic resourcesto the ecophysiology of plant development. The Institut desProductions Végétales merges thesethree groups, i.e. B&PMP, LEPSE(Ecophysiology of Plants underEnvironmental Stresses, see page 26),and BDPPC (Biology of Developmentof Cultivated Perennial Plants, seepage 22). Their presence on the samecampus constitutes a unique opportunity in France to bridge thegap between the genetic, molecularand cellular approaches and the globalexpression in the whole plant in itsenvironment.
The molecular physiology approach is illustrated by the study ofsignals that inform the roots of the nutritional requirements of theaboveground parts of the plant. In this example, an Arabidopsis plantis deficient in nitrogen because half of its root system is placed in a
nitrogen-free environment.The other roots, placed in an environmentcontaining nitrate, are adequately supplied with nitrogen.
Nonetheless, they receive signals indicating deficiency from the aboveground plant structures, and this triggers expression of a geneencoding a specific nitrate transporter.This gene induction is visualizedby hybridization of messenger RNAs in the roots, using a pertinent
probe. Measurements of the uptake of nitrate labelled with the isotope 15N show that the roots' nitrate uptake capacity increases inparallel. So, even though they are not deficient in nitrogen, the roots
adapt their molecular machinery and functioning to the increasednitrogen requirements of the aboveground parts: they respond to
signals whose identification is critical.We can search for the molecularcomponents of such signals indicating nutritional requirements
through functional dissection of the promoters of marker genes ofthis response.
Contact:Alain Gojon, [email protected]
Arabidopsis root-leaf dialogue for better
mineral nutrition
Plant roots aregrown with (left) orwithout (right)nitrates. As answerto a deficiency inthe upper part ofthe plant, a nitratetransporter gene isoverexpressed inthe root
S.Munoz – P.Tillard, © UMR B&PMP
Genes… for which proteins?In molecular physiology, the first step after cloning of a gene isthe identification of the molecular function of the gene product.This is achieved through biochemical and/or physiologicalapproaches which are generally based on the expression of thegene in a heterologous system. For example, the encoded protein may be produced in the bacterium Escherichia coli or ina yeast. In the case of proteins with a role as ion and watertransporters, expression in Xenopus (tropical toad) oöcyteallows direct measurement of the function of these channels.Xenopus oöcyte, like the COS cells of cercopithecus or the Sf9cells of lepidoptera, also constitute a system of expressioncompatible with patch-clamp techniques used in electrophysiological characterization of ion channels.This combination of molecular and physiological tools not onlyallows identification of the function of the gene product, butcan also be used to dissect structure-function relations, usingsite-directed mutagenesis.Contact: Hervé Sentenac, [email protected]
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The proteomics platformof the B&PMP researchgroup (see page 16 “TheMontpellier Languedoc-Roussillon Génopole”) isoriented towards high-throughput functionalanalysis of proteomes:- 2D mapping and construction of databases,analysis of gene expressionprofiles;- robotized identificationof proteins using peptide-mass maps(MALDI-TOF MS),sequencing (ESI MS/MS,sequencer).The B&PMP researchgroup is constructingreference databases,analysing post-translationalmodifications, identifyingresponses to
environmental constraints, and developing new methodologiesfor the analysis of membrane proteomes.These technologies can be applied, for instance, to the adaptivechanges in root morphogenesis induced by mineral deficiencies.Global genomic approaches are used to analyze the changes inArabidopsis gene expression induced by phosphate deficiency.Transcriptome analysis uses medium- and high-density DNAarrays and proteome analysis employs MALDI-TOF technology.These methods are coupled to image analysis of the dynamics ofroot development.This programme is conducted in collaborationwith the Ecophysiology of Plants under Environmental Stressesresearch group (see p. 26).The objectives are:- identification of the main physiological functions contributingto adaptive morphogenesis, by identification of groups of recruited genes;- screening for candidate genes involved in the regulation ofadaptive responses;- separation of general and specific pathways of the response toapplied stress (e.g. general response to stress vs. specific responseto phosphate deficiency).Contact: Michel Rossignol, [email protected]
The proteomics platform
Heterologous expression system
Upper left: 2D electrophoresis gel ofArabidopsis thaliana root proteins.
Upper right: bio-informatic analysisof the root protein content during
establishment of phosphate deficiency. Bottom: mass spectrum
of a polypeptide digestion in 2D electrophoresis (MaldiTof)
J.-B. Thibaut, © UMR B&PMP
P.Doumas, M.Rossignol, V.Santoni, N.Sommerer©UMR B&PMP
Hevea is an exclusive source of natural rubber.The homogeneityand productivity of hevea plantations are reduced not only by
the genetic heterogeneity of seed stocks but also by thephysiological ageing of the stocks of selected genotypes.Somatic embryogenesis is used for the rapid and reliable
multiplication of these genotypes.The rejuvenation of plantmaterial and the cloning of the whole tree should enhance
vigour and increase the homogeneity of plantations.Different lines of "embryogenic calli" (cells kept proliferating in
a suitable culture medium) from the PB 260 genotype wereused in the experimental production of 20,000 vitroplants
between 1996 and 2000.The process is now being adapted toother selected genotypes. Since 1992, field trials have been
conducted regularly at five sites (Africa,Asia, Latin America) tomeasure the growth, production and evaluate other agronomic
characteristics of this new plantation material.Contact: Ludovic Lardet, [email protected]
Thousands of rubbertrees born in
the laboratory
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Development and optimizedexploitation of treesThe primary goal of the "Biology ofDevelopment of Cultivated PerennialPlants" (BDPPC) research group is toenhance understanding of the development of ligneous plants usingtemperate and tropical species, inorder to master their functioning,their genetic usefulness, and theiragronomic utilization.•••
he biology of plant developmentembraces knowledge of all themolecular, cellular and
structural processes leading from thezygote to the reproductively viableadult plant (embryogenesis, juvenileand adult development phases).In cultivated perennial species,account should be taken of specificfeatures linked to the perenniality ofcrops, the length of the juvenilephase, the process of lignification,and establishment of a perennialplant architecture.
Perennial plants are subject toenvironmental changes and to
competition in cultivatedpopulations, and adapt to
these changes through more orless marked alterations in
development. These alterationsmay be accelerated through
genetic improvement.
Team and co-ordinatorThe “Biology of Development of
Cultivated Perennial Plants” researchgroup (UMR BDPPC) comprises 33
researchers from Agro.M, Cirad,INRA, IRD and the University of
Montpellier II.Co-ordinator: Françoise Dosba,
[email protected],fax: +33 (0)4 99 61 26 16
Website:http://www.ensam.inra.fr/arbo/arbo34.htm
Biology of the development of
cultivated perennialplants
T
M.-
P. C
arro
n, ©
Cira
d-C
P Hevea vitroclone in field test(Côte d’Ivoire), clone PB 260,
3 years old
Filao is a tropical tree from Oceania which belongs to theCasuarinaceae. Because of its low nutritional requirements
and remarkable tolerance of drought, the filao plays animportant role in countries of the South in the production of
wood and biomass, the protection of soil against all types oferosion, and the restoration of the fertility of degraded
zones. Filao lives in nitrogen-fixing symbiosis with a nitrogen-fixing microorganism called Frankia.The actinorhizal
nodules or symbiotic roots created by this symbiosis resemble modified lateral roots.The research undertaken at
IRD aims to understand the molecular dialogue between theplant and the microorganism during differentiation and
functioning of the symbiotic roots.The filao genes mediatingsymbiotic rhizogenesis – the formation of these modified
roots – are identified and characterized by means of molecularphysiology and cell biology. Genetic transformation of filao isalso used to characterize its genes.The resulting findings willclarify the mechanisms underpinning the transformation of alateral root into a symbiotic root.They will also suggest howto improve the conditions of symbiotic rhizogenesis in filao.
The identification and characterization of the symbiotic genesof filao will allow us to produce new symbioses that are
more efficient or better adapted to environmental stresses.Contact: Didier Bogusz, [email protected]
Didier Bogusz, © IRD
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The scientific goals of the researchgroup are as follows:- to extend to cultivated perennialplants the tools of structural andfunctional genomics developed withother plants, notably Arabidopsis thaliana and forest species,- to develop the technologies neededfor study of biological mechanismsand breeding (genetic manipulationsand somatic embryogenesis),- to use modelling methodologies tounderstand the temporal and spatialrelations between the developmentand life cycle of perennial plants andthe underlying genetic and molecularmechanisms.
Genes that enable trees to fix nitrogen
Filao symbiotic genes are studiedto improve symbiosis efficiency
Biology of the development of cultivated perennial plants
Eve
lyne
Cos
te,
©IN
RA
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From gene to architectureThis research is currently being pursued at three complementarystructural levels:- molecular: identification of thegenes involved in the different phasesof development of perennial plantsand to investigate the regulation oftheir expression, use these genes tounderstand the architecture of thesespecies with a view to improvement,description of the mechanismsmodulating genome expression. - cellular: development of in vitroregeneration systems to studymorphogenetic responses to environmental changes; identificationof the important stages of growth androle of growth regulators; genetictransformation adapted to perennialplants.- whole plant: root and branch development and architecture inrelation to genotype and environmental conditions, relationsbetween architectural features andthe genetic mechanisms underlyingthe expression and regulation of thegenes concerned.
The perennial species used in thesestudies are:- temperate species: apple tree (seeright "Architecture of trees and qualityof fruits") and vine, and for more specific topics, apricot tree, olive treeand pear tree; - tropical species: oil palm,Casuarina (see left "Genes that allowtrees to fix nitrogen"), coconut palm,coffee and banana, and for more specific topics, hevea (see page 22"Thousands of rubber trees born inthe laboratory"), cacao andEucalyptus.
Trees architecture and fruit quality
The architectural analysis ofplants, developed in the 1960s inintertropical forest, is being appliedto temperate fruit-bearing speciesby the "Architecture andFunctioning of Fruit-bearingSpecies" research group of theINRA and Agro.M at Montpellier.This involves observation of thetree, which is considered globallyand in its temporal context.Thisanalysis allows to reconstitute thehistory of the tree's developmentfrom morphological markers.The main goals of this work are thestudy and analysis of the rulesgoverning early stages of production,and its sustainability of trees of agiven species.
This work leads to propose adaptive management practices
based on in-depth knowledge of the rules governing varietalgrowth and fructification.Against an economic background of overproduction, the production of quality fruits is of particular interest today. Knowledge of the positioning of fruitsin the foliage and the characteristics of the supporting woodwill enhance understanding of fruit quality.This approach takesinto account management practices, notably tree shaping andthinning out the fruits.This multidisciplinary approach bringstogether morphologists, physiologists, ecophysiologists,agronomists and plant biotechnologists from INRA.
The BDPPC research group's primary goal is to study thegenetic determinism of the principal architecturalcomponents. We plan to make genetic improvements on thebasis of enhanced understanding of the mechanisms underpinningthe architecture and functioning of plants of economic or environmental value. Using the apple tree, we shall identify andmanipulate the genes involved in the different developmentstages and in their regulation, notably those involved inmorphogenesis of caules or roots.This will involve the use ofgenetic transformation systems. In the longer term, it will benecessary to integrate into breeding schemes morphological characteristics governing yield and toallow selected material to evolve towards more integrated culture systems that require less input and are less environmentally costly.Contact: Evelyne Costes, [email protected]
Comparison oftree architecture
for two apple treeof the Fuji variety
that have beenmanaged
differently (freegrowth left,
“solaxe” growthright).
Consequence forfruit size
distribution.
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Tolerance of drought:a multifactorialdeterminismResearch on drought tolerance consistsin optimizing the photosynthesis,growth and development of plants soas to maximize production for agiven water supply. The strategy forimprovement cannot be the same asthat followed for other traits, such asresistance to a disease or to a herbicide,which depends on the transfer of oneor a few genes. This is because:� the climate varies year on year andaccording to location, and so a genotype resistant to one droughtscenario will not necessarily be resistant to another. A purely experimental
gricultural production is clearlycontingent on a minimumwater supply: in plants,
photosynthesis and growth are onlypossible if there is transpiration.Carbon dioxide enters leaves throughmicroscopic pores, the stomata,through which water is transpired inlarge quantities (up to three times theplant's weight a day in summer). Ifthe plant cannot take up this amountof water, it must slow its transpirationby closing the stomata and reducingleaf growth. This avoids dehydrationbut also reduces photosynthesis. The"water for carbon dioxide" trade off isinevitable, regardless of any geneticprogress.
The search for better adaptation of plants to waterdeficit is vital for agricultural
production in the decadesahead. Headway can be made
through the use of biotechnology and modelling
of plant gene regulation.
Team and co-ordinatorThe“Laboratory of Ecophysiology of
Plants under Environmental Stresses”research group comprises 13
researchers from Agro.M and INRA.Co-ordinator: François Tardieu,
[email protected],fax: +33 (0)4 67 52 44 43
Creation of drought-resistant
plants?
A
F.Tardieu, © INRA
Natural genetic variability in the response of foliagegrowth rate to water deficit in the air and soil
A model has been established which predicts the responses ofleaf growth rate to soil water status, air humidity, and the temperature of the meristem.This model has been tested bothin the laboratory and in the field, by relating the environmentalconditions to the measured growth rate (photograph).We arenow evaluating the genetic variability of the responses to eachset of environmental conditions for 100 recombinant lines ofmaize.This will enable us to relate gene alleles to each environmental response.Contact: Bertrand Muller, [email protected]
Drought and rate of leaf growth
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evaluation of a genotype wouldrequire dozens of years, which ishardly feasible for practical and economic reasons.� the search for an optimum is moredifficult than the search for a qualitative characteristic, and involvesthe farmer's strategy. When choosinga genotype, the farmer may seek tomaximize the potential yield, therebyrunning a high risk of total loss ofyield, or to maximize protectionagainst risks and therefore settle for alower potential yield.
The LEPSE (Laboratory forEcophysiology of Plants underEnvironmental Stresses) seeks toconstruct and evaluate genotypesexhibiting contrasting responses toseveral water deficit scenarios, from aweak response (maintenance ofphotosynthesis, growth and transpiration: maximized potentialyield at the price of a higher risk) to astrong response (better protection,lower risk, reduced potential yield).To achieve this, we combine modellingwith screening for genes of interest. Afour-pronged approach is used:
� Exploration of the natural geneticvariability in the responses ofgrowth and development to waterdeficit in the soil and air (maize,Arabidopsis thaliana, Dactylis). Thecombination of development models,micrometeorological measurementsand genetic analysis (search forQuantitative Trait Loci) allows individual identification of the allelescharacteristic of the response to temperature, atmospheric humidity,and water deficit in the soil (collaboration with the INRA geneticsgroups at Moulon and Versailles).Similar research is exploring genotypic variability in the capacityof Dactylis to survive severe watershortage. •••
Modelling the effect of a genetic transformation ontranspiration and the water status of plants subject towater shortage
Nicotiana plumbaginifolia plantshave been transformed at agene involved in the synthesisof abscissic acid (ABA).Theresulting enzyme-deficientplants lose all capacity toclose their stomata. Partiallyenzyme-deficient plants weresubjected to different waterdeficits or were grafted ontowild relatives or fed artificialABA.All behaviours of thetransformants under all theconditions tested can be predicted by means of a singlemodel (see picture), whichvaries only in the rate of synthesis of ABA.Contact:Thierry Simonneau,[email protected]
Predicting the effects of transgenesis
F. Tardieu et T. Simonneau, © INRA
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� Analysis of the expression of genesassociated with adaptations inplants subject to known environmental conditions (maize,Arabidopsis, Dactylis)
Gene expression analysis can now bedone in the field or laboratory, thanksto advances in micrometeorologicalmeasurements. In collaboration withthe B&BMP research group (see page18), we assess mRNA (cDNA microarrays) and proteins (Westernblots, analysis of activity) to establishcorrespondences between geneexpression and a quantitative variablein different environmental scenarios(for example, expression of a cellcycle gene and the rate of cell division).
� Systematic assessment of theeffects of a gene manipulation onthe genotype's responses to environmental conditions
Because the plant is a regulated system,several variables characterizing thephenotype are functionally related.Modelling reduces the apparent complexity of the responses to theessential characteristics of eachgenotype. The main test consisted inthe manipulation of a gene involvedin the synthesis of one hormone thatcontrols transpiration and growth inplants subject to water shortage.
�Modelling of the behaviour of agiven genotype in a large number ofpedoclimatic scenarios (Arabidopsis,sunflower)
The microclimatic characteristicsand genotypic traits are fed into acomputer in order to calculate wateruptake, biomass production, etc. Thisoperation has a low marginal cost,therefore it is possible to simulate alarge number of scenarios and calculate the risks associated with agenotype. This work is in its earlystages, and as yet we cannot establisha correspondence between one geneand a simulation of yield. However, itis already possible to establish thiscorrespondence for the simplestfunctions, such as transpiration or therate of foliage growth (in collaborationwith the Cirad-INRA-AMAP unit).
©H
. R
ey,
Cira
d-A
MA
Pet
J.
Leco
eur,
INR
A
Three-dimensional computerrepresentations of
Arabidopsis thaliana
Creation of drought-resistant plants?
Modelling the architecture of sunflower and Arabidopsisthaliana subjected to environmental constraints
Computer modelling is essential in simulating the long-termeffects on plant behaviour of stress and/or an allelic variation.Aprogramme is under way to represent in real time the adaptiveresponses of sunflower and Arabidopsis genotypes to transient
variations in environmental conditions. In Arabidopsis, severalecotypes have been systematically compared for the architecture
model parameters.Contacts: Jérémie Lecoeur, [email protected], Hervé
Rey, [email protected]
Virtual plants in simulating resistance
to stress
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Blast damageevaluation in a
rice trial, with aresistant variety(center row) and
susceptible varieties (side
rows)
J.-L. Notteghem, © INRA
allow progress in: - population genetics,- genetics of interactions,- mathematical modelling of population fluctuations. •••
he Biology and Genetics ofPlant-Parasite Interactions forIntegrated Control (BGPI)
research group is developing novelapproaches to integrated pest management through improvedcombinations of biocontrol methods.We aim to enhance understanding of plant-parasite interactions interms of the underlying mechanismsand at the population level. Basicresearch on suitable models are yielding approaches and tools that
Ever-increasing consumerdemands for quality food and
preservation of the environment mean that
solutions must be found to thenew constraints on agriculture.
It is crucial to developmethods that limit the use of
phytosanitary treatments, andto encourage use of alternative
approaches such as varietalresistance to diseases and
pests. These research activitiesshould be paralleled by
development of decision-making aids.
Team and co-ordinatorThe “Biology and Genetics of Plant-Parasite Interactions for IntegratedProtection” research group (UMR
BGPI) comprises 25 researchers fromAgro.M, Cirad and INRA.
Co-ordinator: Jean-Loup Notteghem,[email protected] fax: +33 (0)4 67 54 59 77
Plant-parasite interactions prospects for integrated pest management
T
Rice blast caused by Magnaporthe grisea is the most importantfungal disease of rice, and greatly limits production, particularlyin Latin America.The resistance of a new variety is in generalquickly overcome by the pathogen, and the cost of fungicides isprohibitive for most rice growers.Rice blast is a research model for numerous internationalresearch teams.A large collection of isolates has been assembledand is used in studies of M. grisea population structures andtheir evolution.This work has been conducted at differentlevels: worldwide, European, Chinese and fertile populations ofAsia.The mechanisms of interaction have also been studied. Newresistance and virulence genes have been mapped. In a collaboration with an Aventis-CNRS (National Centre forScientific Research) research group, one of the avirulence geneshas been cloned, and others are now being studied.The cloningof a resistance gene is currently under way. Genomics is nowbeing used to identify and clone genes involved in the defencemechanisms of rice.Contact: Didier Tharreau, [email protected]
The Rice-Magnaporthegrisea pathosystem asa research model
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Red leaf mottle
in sugarcane
induced by the
Peanut Clump
Virus (PCV)
P. B
audi
n, ©
Cira
d-C
A
Peanut ClumpVirus (PCV)infecting sugarcane
Senecio inaequidens, the narrow-leaved ragwort, an exoticweed which invades natural areas and crops, such as the vine
in Languedoc-Roussillon, is expanding throughout Europe.This species is sensitive to a rust fungus from Australia,
Puccinia lagenophorae, which reached Francesome thirty years ago.We are studying the
epidemiology, population dynamics, and geneticstructure of narrow-leaved ragwort, and the
ragwort's interaction with the rust fungus, witha view to establishing whether the rust fungus
could be used as a biocontrol agent.This project strengthens ongoing collaborations of the BGPI
research group with associate members of Agropolis andwith local laboratories of Australian (CSIRO1) and American(EBCL-USDA-ARS2) organizations specialized in biocontrol
(1) CSIRO: Commonwealth Scientific and Industrial Research Organization (2) EBCL: European Biological Control Laboratory - USDA-ARS: United States Department of Agriculture -
Agricultural Research Services
Contact: Jacques Maillet,Agro-Montpellier,[email protected]
Diagnosing viral diseasesThe diagnosis of viral diseases is a major priority which calls for
aetiology and pathogen characterization. Cirad is developing tests forthe serological or molecular detection of a large number of
microorganisms that attack cultures of banana, sugar cane, cacao,vegetable crops, palm trees including coconut palms. New detectionmethods are constantly reviewed to guarantee the quality of Cirad's
phytosanitary testing in international quarantine services for sugarcane and the indexing of banana (VIC-INIBAP1), taking into accountthe risk of emergence of new viral strains, or evennew pathogens.Through these studies we hope to
develop diagnostic kits for onsite use by our partners.
(1) VIC-INIBAP:Virus Indexing Center of the International Network for the Improvementof Banana and Plantain
Contact : Michel Peterschmitt,Cirad,[email protected]
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Narrow-leaved ragwort-rust interactions to
develop biological controlmethods
Our group is pursuing three lines of research:
• In studying the mechanisms ofinteraction, we use gene mappingand cloning to analyze the pathogenicity of the agents thatcause plant diseases, as well as thedisease resistance of plants. Ourstudy of the genomics of the defencereactions of rice is based on the plantmaterials produced by the teams ofthe Montpellier Génopole (see page 16).
• The study of the factors of viralepidemics transmitted by insects isbased on a space-time study of theepidemics and an analysis of themajor factors: vector biology, vection,diversity of viral populations, hostresistance, geographical fragmentation of cultivated regions,insect control methods. This work isbeing done in close collaborationwith farmers and technical institutesspecializing in this field.
• The study of the population genetics of phytoparasitic fungi,bacteria and nematodes, and oftheir interactions with resistant cultivars has been boosted by recentdevelopments, such as the use ofmolecular tools to characterizepathogen populations and theirmechanisms of genetic evolution.Plant-pathogen interactions areinvestigated in terms of the effect ofhost resistance on pathogen population structures, pathogenicity,and plant resistance.
Applications of the research projectsinclude diagnosis of plant diseasesand expertise on the sustainabilityand optimal management of resistance. Characterized resistancesare used by the plant geneticists andbreeders. Methods are proposed tolimit vector transmission of viralinfection.
Damage causedby Puccinialagenephorae(rust) on narrow-leavedragwort
Jacq
ues
Mai
llet,
©A
gro.
M
M.
Cha
tene
t, ©
Cira
d-C
A
Dissemination of innovations
From research to applications
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In vitro culture allows routinepreparation of thousands of"certified copies" of a plant froma simple tissue fragment fromthe mother plant.This is achievedthrough the use of complexmixtures of mineral salts, sugars,amino acids, vitamins, and
growth regulators. Although a liquid medium is consideredideal for the mass production of vitroplants, there are frequentproblems of hyperhydricity and of physiological disorder(asphyxia) caused by the presence of residues in the medium.It has long been known that temporary immersion reducesthese problems. From 1988, the Biotrop laboratory of Ciradhas worked on the use of this technique and the developmentof a simple, easy-to-use apparatus: Rita (automated temporary
immersion recipient).This two-compartment apparatus makesuse of the advantages of the liquid medium but does not sufferits drawbacks, since the plant material is only briefly in contactwith the liquid. Rita is easy to use because of its design and sizeand is currently being utilized to micropropagate a large numberof plants, notably selected hybrids of Coffea arabica.Contact: Marc Berthouly, Cirad, [email protected],UMR BDPPC (see page 22)Website: http://www.cirad.fr/produits/rita/fr/accueil.htm
Facilitating the creation of newvarietiesThe creation of a new variety is time-consuming and costly.Numerous techniques now improvethe performance of traditionalmethods, and some of these are presented below.
• Haploid methodsThe use of pure lines is often necessary when the goal of the breeding programmes is to createhybrids. Traditional methods needeight to ten years, whereas haploidmethods (haploid-diploidization)rapidly yield hybrid varieties by generating pure lines in a singlegeneration. Plants with only half ofthe genetic make-up are produced bygrowing reproductive cells – ovules orpollen – whose genetic make-up is
then doubled to restore fertility. Forinstance, a cross between such purelines is currently used to producehybrids of tropical or temperate rice.
• Somatic fusions and hybridizationsThe exchange of genetic material between different varieties duringreproduction is an important sourceof variability, but some species hybridize poorly and are thereforedifficult to improve by classicalmethods. Somatic fusions and hybridizations enable mixing in thelaboratory of the genetic and cytoplasmic structures of plants byfusing protoplasts, thereby increasingthe genetic diversity of certain species. At Agropolis, this techniqueis used with citrus fruits.
• Marker-assisted selectionTo ensure that a cultivated hybrid hasrecovered the gene or genes controlling the agronomic trait of
The RITA device with coffee vitroclone
RITA, an apparatus whichfacilitates in vitro culture
Marc Lartaud (© Cirad-Amis, Biotrop)
Improved seeds and plants arethe traditional and most
favoured way of transferringgenetic advances to producers.
Plant biotechnologies havegreatly accelerated and
enhanced this transfer. Theresearch groups of the
Languedoc-Roussillon regionparticipate actively in thedevelopment of these new
technologies. They have fine-tuned methods and
processes that are availablefrom Agropolis, and some of
these have been transferred tothe private sector and the
countries of the South.
Vitropic S.A., a subsidiary of Cirad, isa laboratory at St Mathieu deTréviers near Montpellier which produces vitroplants (plants obtainedby in vitro culture). It was set up in1986 and now has an annual turnoverof 1 million US$, 15 employees, andproduces two to three million vitroplants a year, especially of banana, which are sent mainly to theWest Indies and Africa.Vitropic is aleader in these foreign markets andits planlets production site is one ofthe more important in the world forbanana.An example of how biotechnology improves productionThe production of vitroplants doesnot involve genetic transformationtechniques but rather tissue culture.
Plants selected for their agronomic traits are multiplied rapidlyin test tubes.The miniature banana plantlets produced underthe protected laboratory conditions are disease-free and areshipped from Montpellier to the production zones.Their utilization as planting material enhances production, since vitroplants give greater yields with less use of pesticide whengrown in soil free of nematodes (soil parasites). In the case ofbanana, vitroplants help promoting a more environmentally-friendly agriculture.The need for constant innovationTo increase its know-how and to improve and diversify its products,Vitropic must innovate constantly and invest inresearch and development.Agropolis, and in particular Cirad,provide Vitropic with a favourable environment and expertscientific and technical partners in the pursuit of its goals.Vitropic operates mostly in the Languedoc-Roussillon region,but is also developing collaborations with numerous partnersfrom countries of the South.Contact: François Cote, Cirad, [email protected]
Vitropic: a laboratory for production ofvitroplants of tropical species inLanguedoc-Roussillon
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interest, difficult and often time-consuming field tests were traditionally necessary. Moleculargenetics can now be used to identifyand locate genes and to follow themduring crossing and selectionthrough their association with molecular markers. This techniqueallows concentration of the mostvaluable traits and avoids their lossduring these operations. This processwas first applied to simple characteristics, and is now increasinglyused for quantitative traits, which aremore difficult to handle.
• Direct transfer of genes by transgenesisThe transfer of an agronomic trait toa hybrid is particularly lengthy anddifficult by natural means, but it isnow possible to transfer the genecontrolling the relevant characteristicdirectly into the cultivated plant bymeans of transgenesis.The first applications of this methodare much debated in Europe, yet atthis early stage much remains to bediscovered. Among the varietiesselected, increasing use is made oftransfer of a valuable trait to a plantof the same or a similar species. •••
Use of bananavitroclones
increases production with
less pesticides
Yva
n M
atth
ieu,
©V
itrop
ic S
.A.
Multiplying the most interestingpalm tree in large numbers to fix
useful agronomical characteristics
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In vitro culture to produce true copiesof oil palm The oil palm is the major source of vegetable fat in the humidintertropical zone.With an annual output of ca. 20 millionstons, it accounts for 20% of world oilseed production.Conventional techniques of vegetative propagation (cuttings,shoots, grafts) do not exist for this plant with its obligate cross-pollination and seed-only propagation.The mode ofreproduction and the time needed for genetic improvementlead to substantial heterogeneity in the plants grown fromselected seeds available to growers. Because of this, the mostuseful agronomic traits can only be fi xed by means of control-led artificial vegetative propagation using cell biology techniques
ensuring large-scale production of true copies of reproductionof the most valuable plants. Large-scale multiplication and regeneration using somatic embryogenesis have been developedsince the early 1980s in collaboration between IRD and Cirad,and have been transferred to the main producing countries(Côte d’Ivoire, Malaysia, Indonesia) for pilot testing. Several hundred thousand oil palm plantlets regenerated in vitro havebeen planted, and various clones have been identified, leading toa 20 to 30% yield increase, thus underscoring the value of thisapproach.We have also noted the existence of sterile, off-typesor variants, resulting from regeneration.This phenomenon isknown as somaclonal variation and is now being studied indepth in tandem with research teams from Great Britain andMalaysia, using molecular approaches based on cutting-edgetechniques available at Agropolis, notably through theMontpellier Génopole (see page 16).Contact:Yves Duval, Cirad-IRD, [email protected],UMR BDPPC (see page 22)
Yve
s D
uval
, ©
IRD
Multiplication techniques In vitro culture of plants in a sterilenutrient medium is a longstandingand widespread technique in microcutting and micrografting.Other techniques are being improved,including:
• Embryo rescueSimilar but poorly compatible speciesmay sometimes be crossed, but theresulting embryo is often unviable ordoes not develop beyond a certainstage. Yet this type of cross wouldimprove cultivated varieties. Embryorescue allows the production ofhybrids from the first cells of a crossbetween "incompatible" species.Embryos can be rescued at an earlystage, soon after fertilization, cultivated and thus saved. This technique has been used with citrusfruits and rice.
• Somatic embryogenesisThis form of vegetative propagationyields a large number of plantletsgenetically identical to the motherplant that gave the explants. It can beused to reproduce millions of identicalcopies of a plant from a fragment ofleaf or stem. This is a highly promisingtechnique as it allows enormous multiplication rates. It is also usablein transgenic systems. It is especiallyuseful when the plant does not naturally propagate vegetatively (oilpalm, coconut palm...). Despite certain technical difficulties, hundredsof thousands of palm trees, heveatrees, cotton plants and bananaplantlets have been produced by thisprocedure at Montpellier.
Appropriate and officially approvednew equipment for all these techniques is needed, and has beeninvented or adapted by the researchteams at Agropolis. An example isRITA (see page 32 "Rita, an apparatuswhich facilitates in vitro culture"),which was designed to improve the
growth of in vitro cultures. Otherequipment has been developed incollaboration with small- andmedium-sized local companies, suchas confinement greenhouses and culture rooms, several of which havebeen built in Africa and Asia.Agropolis member institutions possess considerable know-how inthis area, particularly regarding invitro cultures, which have a widerange of potential applications.Co-ordinator: Jacques Meunier, Cirad,[email protected]
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AbbreviationsAgro.M: National Agronomy School at Montpellier
DAA: Degree in Advanced Agronomy DEA: Degree of Higher Studies
DESS: Degree of Specialized Higher StudiesDEUG: Degree of General University Studies
INA-PG: National Agronomic Institute – Paris GrignonINH: National Institute of Horticulture (Angers)
UM II: University of Montpellier IIUP: University of Perpignan
Level Degree Courses Institute Director
Bac + 3Under-
graduateBSc
Biology:Cell biology and applied plant
physiologyUM II Yves Sauvaires
Bac + 4
Bac + 5
Bac + 5à 8
Post-graduate
Post-graduate
professionallyoriented
postgraduatedegrees
Doctoral studies
MSc
Special modules
DESS
DAA
DEA (bac+5)
Doctorat (bac+8)
Cell biology and physiology:Applied plant physiology
Conservation of biodiversity:genetic diversity of organisms
The impact of biotechnologieson the seed and breeding
sectors
Regulating agriculture: exampleof integrated pest management
Genetics, genomics andadvanced plant biotechnology
Plant production engineering:seeds and plants
Plant genetic resources andbiological interactions
Plant development and adaptation
Evolution and ecology
UM II
Agro.M
Agro.M
Agro.M
UM II
Francine Casse
André Charrier
Dominique This
Jean-LoupNotteghem
Michel Lebrun
INA-PG / Agro.M / INH
UM II / Agro.M
UM II / UP /Agro.M
UM II / Agro.M
André Charrier
J.-L. NotteghemIsabelle Oliviéri
Michel LebrunMichel DelsenyClaude Grignon
Bernard DelayJacques Maillet
Education and trainingat Agropolis
Agropolis is one of the major French and European training polein agriculture, agribusiness and rural development. The
Universities of Montpellier II and of Perpignan, as well as Agro ofMontpellier (Agro.M), offer numerous courses in plant genomics,
genetic resources and biotechnology.
Degree courses
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The evolving French higher-education system
The reform of the French higher educationsystem begun in late 1998 is intended to harmonize teaching and training within theEuropean area; it emphasizes on three maindegrees: Licence (BSc level), Master (MSclevel) and Doctorat (PhD level). Postgraduatestudents have to choose between a short termcurriculum, professionally oriented orresearch oriented doctoral studies.
Continuing education� Customized trainingAgropolis can organize specific educational events tailored to collective and individual requirements, such as seminars, laboratorywork, on-field training, training of trainers, and so forth.Depending on demand and content, these courses will take placein or outside Montpellier.For further information on personalized training, see our website:http://www.agropolis.fr/formation/servicesfc.htmlPlease note that modules from doctoral studies and professionally oriented degree can be followed in continuing education.
� " Introduction to molecular biology ", one-week courseopen to students with the baccalaureate plus two years ofhigher-education training. Site:Agro.M campus.Director: Philippe Joudrier, [email protected]
Training in molecular biology at Montpellier
Alain Rival, © IRD-Cirad-CP
French system International system
Baccalauréat End of high school
DEUG
Licence
Maîtrise
DESS DEA
Doctorat
Bac+1 years+2 years
+3 years
+4 years
+5 years
+6 years+7 years+8 years
BSc
MSc
PhD
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The Graduate School (GS) embraces all lecture and practicalcourses that lead to the PhD degree.This course begins in theyear of the DEA (baccalauréat+5) and continues with additionaltraining involving seminars, scientific conferences and trainingmodules for the three years of thesis preparation.These moduleshave the twofold purpose of improving the students' scientifictraining and preparing them for their professional career.
The Graduate School “Integrative Biology” at the University ofMontpellier II focuses on the integration in the biological processes and the interactions of biological systems, with a viewto conservation of biodiversity, protection of the environment,sustainable development and agriculture and the progress in biotechnology.The school's six scientific priorities are:
• Biodiversity, mechanisms of evolution, genetic resources.• Genomics: structure and organization of the genome, genefunction and regulation, functional genomics.• Host-parasite interactions and symbiotic hosts.• Ecology of populations, communities and ecosystems; globalchanges.• Physiology and ecophysiology of relations between plants andthe biotic and abiotic environment.• Mathematics and information technology applied to biologicalsystems and processes.
The Graduate School “Integrative Biology” offers seven coursesleading to a DEA degree, with an annual intake of 75-80 students.Some 60 graduates begin the PhD course every year, and thereare almost 200 PhD students in all.The PhD students work insome thirty research groups in universities (UM II / Agro.M) andresearch institutes (Cirad, CNRS, INRA, IRD at Montpellier).
Three doctoral courses are more specifically oriented towardsplant biotechnologies, genomics and genetic resources:
- DEA " Plant Genetic Resources and Biological Interactions"- DEA "Development and Adaptation of Plants" - DEA "Evolution and Ecology".
Graduate school "Integrative Biology"
Agropolis provides information and guidance for foreign studentsand trainees on:- how to obtain a resident or student permit,- finding accommodation,- applying for housing benefit,- campus facilities and restaurants, public transport, culturalactivities…
For certain specialized courses,Agropolis also offers a personalized set of services for foreign students. Depending onthe course chosen, an introductory period may comprise:- French courses,- laboratory training,- additional required courses defined with the training advisor,- personal tutoring.
Some figures…- Number of thesis completed in plant biotechnologies, genomicsand genetic resources: 20-25 per year.- Student population: 75 PhD students, 30 DEA, 15 DESS, 15DAA, i.e. a total of 135 students on various postgraduate courses.
Welcoming foreign students and trainees
Training in a vine collection (Agro.M)
J.-L. Porreye, © Agro.M
Contact- DEA and Graduate School “Integrative Biology”:
André Charrier,Agro.M, [email protected]: +33 (0) 4 67 04 54 15
- Continuing education and services to foreign studentsand trainees: http://www.agropolis.fr, heading "training",
[email protected],fax: +33 (0)4 67 04 75 99
This publication was supportedby the French government and
the Languedoc-Roussillon regional authority
Agropolis member organizationsinvolved in genetic resources,genomics, and biotechnology
research:AGRO Montpellier (Ecole Nationale
Supérieure Agronomique de Montpellier)2, Place Pierre Viala
F-34060 Montpellier Cedex 1Tél : +33 (0)4.99.61.22.00
http : //www.ensam.inra.fr
CIRAD (Centre de coopération internationaleen recherche agronomique pour le
développement)Avenue Agropolis
F-34398 Montpellier Cedex 5Tél : +33 (0) 4 67 61 58 00
http : //www.cirad.fr
CNRS (Centre National de la Recherche Scientifique)
Route de MendeF-34293 Montpellier Cedex 5
Tél.: +33 (0)4 67 61 34 34http://www.dr13.cnrs.fr/
INRA (Institut National de la Recherche Agronomique)
2, Place Pierre VialaF-34060 Montpellier Cedex 1
Tél : +33 (0) 4 99 61 22 00http : //www.ensam.inra.fr
IRD (Institut de Recherche pour leDéveloppement)
911, avenue Agropolis - BP 5045F-34032 Montpellier Cedex 1
Tél : +33 (0) 4 67 41 61 00http : //www.mpl.orstom.fr
Université Montpellier II (UM II)Place Eugène Bataillon
F-34095 Montpellier Cedex 5Tél : +33 (0) 4 67 14 30 30
http : //www.univ-montp.fr
Université de Perpignan52, Avenue de Villeneuve
F-66860 Perpignan CedexTél : +33 (0) 4 68 66 20 00
http : //www.univ-perp.fr
Director in chief :Michel de Nucé de Lamothe
Technical editors : Véronique Molénat, Yves Savidan
Scientific coordinator :André Charrier
Participed to this issue :Marc Berthouly, André Bervillé, Didier Bogusz,
Fabien Boulier, Jean-François Briat, Richard Cooke,Evelyne Coste, François Cote, Michel Delseny,
Martine Devic, Angélique D’Hont, FrançoiseDosba, Stéphane Dussert, Yves Duval, Manuel
Echeverria, Alain Ghesquière, Marc Giband, Jean-Christophe Glaszmann, Alain Gojon, Claude
Grignon, Serge Hamon, Emmanuel Guiderdoni,Philippe Joudrier, Pierre Lagoda, Ludovic Lardet,
Jérémie Lecoeur, Jacques Maillet, Jacques Meunier,Yves Meyer, Bertrand Muller, Jean-Loup
Notteghem, Michel Peterschmitt, Jean-Claude Prot,Hervé Rey, Michel Rossignol, Marc Seguin, HervéSentenac, Thierry Simonneau, François Tardieu,
Didier Tharreau, Patrice This.
Translation : David Marsh
Corrections : M.-C. Kohler , Yves Savidan
Photo acknowledgements : Bernard Marin,Danièle Cavanna, Sémiha Cebti,
Jean-Pierre Grouzis, Thierry Lacombe, Marc Lartaud,
Production :Design Publicis Méditerranée
Impression : IMP’ACT imprimerie
Institut de recherchepour le développement
AGROPOLISAvenue Agropolis
F-34394 Montpellier Cedex 5France
Phone : +33 (0)4 67 04 75 75 - Fax : +33 (0)4 67 04 75 99e-mail : [email protected] http://www.agropolis.fr
