.v. •.._...••• -.. •••..---- .. ----•.. ••• •••BIOTECHNOLOGIES••
Oil palm biotechnology: progress and prospects*
Alain RIVAL " [arnes TREGEAR 2, Estelle JALlGOT 2, Fabienne MORCILLO 2, Frederique ABERLENC 2, Norbert BILLOTIE " Frederique RICHAUD 2, Thierry BEULE 2, Alain,'BORGEL 2, Yves DUVAL 2
I Cirad-CP. TA80/PSfll, bd de la Lironde, 34398 Montpellier Cedex OS, France
Abstract: Today, a range ofbiotechnological approaches, from somatic Resume: Les strategies d'amelioration genetique dupalmier ahuile (Elaeis embryogenesis tobiomolecular research, play anincreasingly important role guineensis jacq.) font desormais appel atout un ensemble d'approches in breeding strategies for oil palm (Elaeis guineensis jacq.). biotechnologiques, allant deI'embryogenese somatique ala biologie mole Clonal micropropagation. Methods ofcloning byinvitro culture led tothe culaire. development ofa micropropagation technique for oil palm based onsoma Micropropagation clonale. Les methodes de cionage par culture in vitro ticembryogenesis which was tested at thepilot stage onelite genotypes, antconduit audeveloppement d'une technique de micropropagation qui a thus enabling theproduction ofhigh oil yielding clones. This phase allowed etetestee aI'echelle pilote sur des genotypes d'elite, permettant ainsi la the identification of limiting factors associated with scaling-up, with respect production declones hautement producteurs enhuile. Cette phase a revele in particular tothescale ofmass production required tomeet theneeds of uncertain nombre de facteurs limitants lies auchangement d'echelle, en planters and to theproblem ofensuring genetic fidelity in theregenerated particulier les productivites necessaites pour satisfaire la demande des plan plant material. These two concerns led researchers tolook further into the teurs et la fidelite genetique du matetiel vegetal regenere. Ces deux pro underlying physiological and/or molecular mechanisms involved in soma blemes antconduit Jes chercheurs aanalyser les mecaaisme: physiologiques tic embryogenesis andthesomaclonal variation events induced bythein et/ou moiecutaues impliques dons I'embryogenese somatique et les varia vitro cloning procedure. tions somaclonales induites parle clonage in vitro. Structural and functional genomics. Marker-assisted breeding in oil palm Cenomique structurale et fonctionnelle. La selection assistee par mar isa long-term multi-stage project including: molecular analysis of genetic queurs constitue, pour le palmier ahuile, unprojet along terme, articule diversity in both E. guineensis and E. oleifera germplasms; large scale deve sur plusieurs phases: I'analyse moleculaire de ladiversite genetique dons lopment of PCR-based microsatellite markers; and parallel development of le germplasm d' E. guineensis et d' E. oleifera ; le deveicppemen: agrande three genome mapping and QTL detection projects studying key agrono echelle par PCR de marqueurs microsatellites, et le developpement enparal mic characters. lele de trois projets de cartographie genetique etde detection de QTLs s'adres Post-genomics. In order to tackle theproblem ofthemantled flowering sant ades coroaeres agronomiques majeurs. abnormality, which isinduced during themicropropagation process, stu Post-genomique. Afin de comprendre le phenomene d'anomalie florale « mant dies ofgene expression have been carried outin tissue cultures as a means led ", induite aucours du procMe demicropropagation, des etudes sur l'ex ofestablishing anearly clonal conformity testing procedure. It isimportant pression du genome ant ete menees sur des cultures de tissus, dons lebut de toassess what kind ofmethodology is the most appropriate for clonal confor mettre aupoint untest de conformite preaxe. 11 importe d'evaluer quelle mity testing bycomparing RNA, protein and DNA (PCR) based approaches. methodologie estla plus adequate pour cetest, en comparant des approches Parallel studies ongenomic DNA methylation changes induced by tissue basees sur I'analyse des ARN, des proteines ou de I'AON (PCR). Les recherches culture suggest that thelatter may play animportant role in thedetermi menees en parallele sur les changements de niveaux de methylation de I'AON nation ofthemantled abnormality. genomique induits par culture in vitro suggerent une implication decisive de cephenomene dons le detetmimsme de I'anomalie « mantled ».
Key words: Elaeis guineensis jacq., epigenetics, genomics, marker-assis Mats cles : Elaeis guineensis jacq., epigenetique, embryogenese somatique, tedbreeding, molecular markers, somatic embryogenesis. genomique, marqueurs maecutoues, selection assistee par marqueurs.
* Results presented in the present article arepart of anoralcommunication givenat PIPOC 2001 (MPOB International Palm Oil Congress), 20-23August 2001, Kuala Lumpur, Malaysia.
OCL VOL. 8 W 4 JUILLETJAOOT 2001 295 .....
Challenges in oïl seeds by c10ning the best male parents (pisi duction scheme currently used at Cirad/IRD palm biotechnology fera), since pollen production can be alimiting involves 4 distinct stages, as shown in Figure 1. factor [S, 6]; To date, embryogenic suspensions have been - the exploitation of (E. guineensis x E. o/eifera) successfully isolated for more than 20 different Due to the increasing demand for palm oil, the interspecific hybrids, a limited number of which c1onallines. The average concentration was lack of plantable land and the foreseeable are fertile, but which can show agood tolerance found to be ca 105 cell c1usters per litre with a increases in cultivation costs, it is necessary to to pests and diseases, notably in South America multiplication factor reaching 4x per month. make available to the planters planting mate [7]; These characteristics allow mass propagation. rial with high genetic potential. - the production of biclonal seeds from soma For oil palm, Sondahl (pers. com.) estimated the Nowadays, planting material consists solely of tic embryogenesis-derived parents [5]; price of an encapsulated embryo produced by tenera hybrids (fruits with shell of intermediate - the true-to-type regeneration of future gene bioreactor technology inthe USA at ca 0.20 USD, thickness), originating from crosses between tically-engineered materiel bearing useful agro to which has to be added the cost of in vitro cul dura (thick shell) and pisifera (thin shell) types, nomic traits [8]. ture for germination, rooting and acclimatisa the thickness character being controlled by a The biological characteristics of the oil palm do tion. Field trials are under way for the assess monofactorial gene [1]. not allow its vegetative propagation by conven ment of the clonai fidelity of plantlets originating The breeding strategies developed by seed com tional horticultural means, therefore, the only from cell suspension cultures. The first results panies are aimed at producing dura x pisifera possible way to c10nally propagate elite oil palms obtained from suspension culture-derived clones hybrids with a high productivity of oil contain is by means of somatic embryogenesis. C10ning are quite encouraging, and various research ing ahigh proportion of unsaturated fatty acids, of oil palm (Elaeis guineensis jacq.) is performed groups from the oil palm industry in Malaysia a low growth rate and, in certain cases, resis by inducing somatic embryogenesis on calli deri have now developed embryogenic suspension tance/tolerance to diseases such as vascular wilt ved from various tissue sources, using tissue cul cultures growing on 2,4-0 free or even auxin caused by Fusarium oxysporum elaeidis in Africa ture protocols recently reviewed in detail [8]. free media. These protocols are presently under [2]. Breeding schemes now incorporate the assessment and initial observations of trueness exploitation of genetic resources able to pro Current protocols to-type have revealed agenerally very low pero vide tolerance/resistance to diseases which are centage of abnormal palms. A management stra confined to specifie parts of the ail palm culti Approximately 25 years ago, two major groups tegy, based on the sequential cryopreservation vation area: Ganoderma disease in South East initiated research programmes for oil palm of embryogenic lines [18] cou Id be followed in Asia; and also bud rot in Latin America. micropropagation: in the UK and in Malaysia, order to lower the risk of losing embryogenic Since each selection cycle lasts for around the Unilever Plantations and Harrison and capacity in suspensions during multiplication. 10 years, genetic improvement is very slow, Crossfields Plantations group [9, 10] and in even if much progress has been achieved over France and Côte d'Ivoire, the IRHO/Orstom Embryo maturation the last 50 years [3]. Avery high heterogeneity group (which became Cirad-CP/IRD in the is still observed among hybrids, some palms 80's) [4, 11]. These programmes were initiat ail palm somatic embryos produced from producing 60% more oil th an the average of ed to complement in-house breeding strate embryogenic suspension cultures exhibit an the progeny of agiven cross [4]. These charac gies and were aimed at multiplying the elite incomplete maturation and develop directly teristics must be considered along with the low germplasm available in the plantations for com towards germination without passing through planting density (generally ca 143 palms per mercial use. In a recent review [8] a descrip aquiescent phase [19]. Studies have been car hectare) and the necessity of establishing seed tion was given of different regeneration pro ried out in the aim of understanding the bio orchards for the production of commercial plant tocol applied to oil palms from various different logy of embryonic development and to iden ing material; thus it can be seen that oil palm genetic origins. tify factors involved in maturation, a critica! step improvement is labour intensive, time consum in the determination of somatic embryo qua ing and therefore expensive. Large scale propagation through lityfor several species [20]. Changes in the abun Biotechnological approaches applied to oil palm embryogenic suspension cultures dance of compounds involved in the determi breeding have considerably increased in their nation of desiccation tolerance (such as importance, not only in micropropagation, but Progress has been made in plant production oligosaccharides and abscisic acid [ABA]), or in also in marker-assisted breeding, and more through somatic embryogenesis, particularly by the vigour of regenerated plantlets (such as sto generally, in physiological and molecular stu developing systems based on the artificial seed rage proteins) were investigated. dies of the expression of genes of paramount concept [12] in which the somatic embryogene agronomic value (flowering, abscision, etc). sis process is used to produce individual embryos Acquisition of desiccotion to/eronce with relatively synchronous development. Zygotic embryos of oil palm can withstand com The initiation of embryogenic oil palm cell sus plete desiccation and dehydrated seeds can be Clonai propagation pensions has been reported by several authors stored for two to three years. Dry matter, water [13, 14] thus demonstrating the feasibility of this content, sugar and ABA contents were investi The constraints which exist in oil palm breeding technique for a rather "recalcitrant" model. gated throughout zygotic embryo development make desirable the development of a vegeta Research has been carried out [13, 1S, 16] in order in relation to the acquisition of desiccation tole tive propagation technique which would allow: to develop new methods of large scale micro rance. The acquisition of desiccation tolerance - the exploitation of the variability existing propagation for oil palm (see also areview [17]). between the 3rd and the 4th month was asso among the tenera hybrids by c10ning elite indi ln oil palm, embryogenic suspensions are esta ciated with sugars and ABA synthesis, under viduals [4]; blished from friable, nodular calli, which are iso Iying the role of these compounds in the phe - an increase of the production of high quality lated from nodular compact calli [13]. The pro- nomenon [21, 22]. In Figure 2 are summarised
296 LA FILlËRE AUJOURD'HUI DEMAIN .....
Figure 1. Diagram of oil palm micropropagation through embryogenic suspensions (Cirad-CP/IRD protocol).
PROLIFERATION PRE- TREATEMENT MATURATION GERMINATION
..!.~D iquid medium GE'li(ied mE'dium Gelified medium Active ch~rcoal \1 growlh r 'gulalor
-- t t BAP SUCROSE
4 weeks 6 weeks 8 week
on a single graph the main changes in bioche tic embryos of oil palm, a cDNA clone, GLO7A, These results open the way for the use of stra mical and physiological parameters occurring has been isolated for use as a probe in northern tegies based on the "artificial seeds" concept in planta throughout oil palm embryogenesis. hybridisation studies [26]. for oil palm. The latter could be obtained from Taking these results in account, the effects of ln summary, it is possible to identify markers of embryos produced at ahigh rate from embryo sugars and ABA on desiccation tolerance, soluble maturity (sugars, ABA and storage proteins) in genic suspensions, which would then be cor sugar content and germination rate of oil palm zygotic embryo which can be used to evaluate rectly treated to achieve maturation (enrich soma tic embryos were investigated [22]. The soma tic embryo quality. The results achieved ment in storage proteins). 5ingle soma tic enrichment of the medium with sucrose and on somatic embryo allow to define in vitro cul embryos would be encapsulated and stored ABA improved somatic embryo maturation and ture conditions for improved tolerance to desic (either at room temperature or at 0-4°C) before desiccation tolerance which remains however cation and accumulation of storage proteins. delivery at the appropria te time, according to incomplete as compared to zygotic embryos. Further investigations will be undertaken to the planting season, to tissue culture laborato improve maturation protocols. ries or nurseries situated close to the planta- Accumulation of 5tarage prateins 5torage prateins accumulated during oil palm Figure 2. Changes in biochemicol and physiologicol parameters occurring in planta throughout ail palm embrya embryo develapment were extracted, purified genesis. and characterised [23]. Only water- and low salt-soluble proteins, with respective sedimen tation coefficients of 25 and 75, were detected T__:::-,-,PH-"Ac.::S-=-E-,,1,----_".44_-:-,P-"H-"A-=-SE=:.:I-,,1__" '44_-"Pc.:.H.::.A",S.;:.E.::.111'---_ Ontogenesis ,'-'\aturation Acquisition in mature embryos. Alter purification by gel fil quicscent stail' tration, the various protein classes identified ~_~....---~------Numberoi cells were characterised by electrophoresis and amino acid composition analysis. Ra(finose The accumulation of 75 globulins was studied in zygotic and somatic embryos [24]. The _-i-"I----:;;-:----- Desiccat ion tolerance ~-~----- amounts of soluble protein and 75 globulins in Stnragc proteios somatic embryos were found to increase rapidly during the early stages of development, but were almost 80 times lower than in zygotic embryos. The in vitro production of 75 globulins (and more Water content generally salt-soluble prateins) was impraved by the addition to the culture medium of glu ta mine, arginine, sucrase and ABA, the effects of these components being additive [25]. '---_.--::.. -'-...e:....--'- ... Days aiter To investigate further the regulation of 75 glo o 20 40 60 80 100 120 140 160 180 fertilisation bulin gene expression in both zygotic and soma-
OCL VOL 8 W 4 JUILLETlAOÙT 2001 297 . -.. tions, where germination either in vitro or ex male parts in flowers of both sexes, called the The DNA methylation hypothesis vitro could be carried out. "mantled» phenotype [8, 34). This somaclonal variation phenomenon may result in partial or ln recent years, evidence has been accumulat· Physiology of vitroplants complete rJower sterility, thus directly affecting ing that DNA methylation at the genome wide oil production, depending on the severity of the level plays a key raie in regulating plant deve· Physio/ogy of in vitro rooting abnormality. Interestingly, reversions to the nor lopment [41). Following induction by auxin treatment, the in mal phenotype over time have been found ta Changes in DNA methylation on deoxycytidine vitro rooting performance of oil palm shoots occur, leading ta a complete recovery of the (dC) residues have been shown ta be involved varies considerably. A study [27) has described normal phenotype for 100% of the slightly in the regulation of gene expression at the trans a preliminary evaluation of gaiacol-peroxidase mantled individuals, and for 50% of the seve criptionallevel, particularly during the diffe· activity as a marker of in vitro rooting, which rely mant/ed on es arter 9 years in the field [35). rentiation/dediHerentiation processes and as a has enabled major improvements in the stan Several potential biochemical markers of the mant response to a variety of environmental stresses dard rooting protocol, which initially involved /ed abnormality in oil palm have been investiga [42, 43J. Furthermore, micropropagation pro two separate induction/expression phases. A ted: polypeptide patterns [36) and end age nous tocols often involve growth regulator treat· single rooting step is now used, with an auxin cytokinins content [37) have been studied com ments, which might affect the level of DNA treatment applied over a much longer time paratively in normal and mantled plant material. methylation. Genetic and phenotypic variation (8 weeks) using lower NAA (Naphthalene Acetic Nevertheless, it has been very difficult to assess found among regenerated plants has been term 1 Acid) concentrations (0.5-1.0mg.L- ). This quite the validity of such markers on alarge number of ed somaclonal variation [44). It is now c1ear that long induction/expression phase probably acts sampi es, because of the lack of reproducibility (in a diversity of genetic and epigenetic changes as a buHer stage, which is able to stabilise the the case of proteins) or the high cost (in the case are underlying this instability, which may not physiological status of shoots (peroxidase acti of endogenous cytokinins) of such estimations. be the result of a single causal mechanism [45, vity, levels of endogenous auxins) before the Flow cytometric analyses pedormed on seed 46). inductive treatment by auxin may act. lings and on normal/mantled plant material We firstly chose ta use a global approach for demonstrated a uniform 2C ploidy level [38). the investigation of DNA methylation rate, ln vitro photosynthesis and acclimatisation Extensive Random Amplified Polymorphic DNA aimed at revealing differences between normal Several studies have been conducted in ail palm (RAPD) experiments, involving the examination and variant plant material. Using two genome in order ta reduce acclimatisation losses [28 of 8,900 markers, also failed ta show banding wide quantification methods (HPLC estimation 32). The in vitro photosynthetic parameters of patterns discriminating either the mother palm of total 5 mdC concentrations, and in vitro satu somatic seedlings have been measured throu genome from its clonai offspring, or true-to ration of CG sites with methyl groups by the ghout soma tic embryogenesis-based c10ning type regenerants from somaclonal variants [39). Sssl Methylase-Accepting Assay), we demons procedure, with the aim of characterising the Previous studies have shawn that the mant/ed trated [47) the occurrence of a significant geno physiological status of the in vitro regenerated abnormality is epigenetic in nature. Firstly, it mic hypomethylation in ab normal calli (-4.5%; plants and thus optimising success rates during has been observed that reversion to a normal p < 10-5) and leaves (-1.2%; p < 10-5) from acclimatisation. floral phenotype may occur in the field [8]; "mantled" regenerants, compared with their Ali the studied photosynthetic parameters (pho secondly, although the mantled abnormality is normal counterparts (Figure 3). tochemical activities, CO 2 exchange and car strongly transmitted through tissue culture, only This study on global methylation rates provides boxylase enzymatic activities) indicate that, in aweak non-Mendelian transmission occurs via us with a first glimpse of the molecular changes oil palm, photosynthetic activity could be mea seeds [40). Thirdly, our previous studies did not associated with the mantled abnormality and it sured as early as during the first caulogenesis allow us to identify any major alterations in is consistent with the epigenetic characters step, showing a noticeable increase during the genomic DNA structure that could be linked observed, including reversion. Despite the highly second step. Subsequently, photosynthesis with the mantled phenotype. significant decrease in methylation rate observ- decreased during root growth. With respect ta Grout's classification [33], it can be assumed that the oil palm belongs to the Figure 3. Comporison 01 Global Methylation Rates (%) 01 genomic DNA in mature leaves lrom normal and mant led variants. 7sampled palms/done/type. 3 hydrolyses. Interoction: F(3, 113) = 35.40; P < 0.0000. c1ass of plants in which in vitro-grown leaves can contribute to autotrophy and then play an active part in acclimatisation. It is therefore 32 0 highly probable that acclimatisation losses could v be due principally to poor or incomplete root ,,..,E 30 ~ ing and/or ta a poor management of the envi 2... 21l h ronment of vitroplants (especially concerning ~ b '"c RH) during this very critical step. 0 ;:; 26 >..'" -'= 24 Molecular analysis "'E ~ of somaclonal variation (5 22 l3 Approximately 5% of soma tic embryo-derived 20 oil palms show abnormalities in their rJoral deve lopment, involving an apparent feminisation of
298 LA FILIlORf AUJOURD'HUI DI'MAIN ......
ed in mantled palms, it is likely that very few of abnormality. Most of our efforts to date have material as for the differential display experi the corresponding cytosines play a direct role however been concentrated on investigating ment. Next, markers confirmed as being diffe in the triggering of the somaclonal variant phe whether mantled-dependent gene expression rential are tested on various other cultures to notype. patterns may also exist during the in vitro stages assess whether gene expression is consistently Global DNA hypomethylation associated with of the micropropagation procedure. At the vege associated with montled status. Any markers local genetic or epigenetic defects has already tative stages of development, no distinct mor considered sufficiently promising after this stage been documented in several cases of develop phological criteria are available for screening out are characterised in detail by the isolation of the mental abnormalities, in plants [43] as weil as abnormal palms. The identification of "finger cDNA coding sequence, Northern studies of tis in animais [48]. print" genes displaying montled-related expres sue specificity and genomic 5' flan king region Therefore, we considered it necessary to target sion at pre-planting stages would thus be of isolation. The latter allows to investigate whe more precisely those sequences which, when great benefit by providing us with the means to ther any conserved cis-acting elements are pre misregulated, cou Id potentially account for the formulate molecular tests for clonai conformity. sent in the promoter region which might shed "montled" phenotype, or which could be used Extensive studies have been carried out on gene light on the regulation of the gene in question. as markers for the early detection of DNA methy expression in oil palm tissue cultures as a means An illustration of the results obtained in the lation perturbation in the regeneration process. of identifying putative early markers for clonai various stages of marker isolation and valida- To this end, we have been carrying tion is shown in Table 1. In this case, out methylation-sensitive RFLP and Figure 4. Experimentalstrategy for the identification and characterisation of genes data obtained for leafy shoot mate AFLP studies, involving the isoschi displaying a mantled-dependent expression pattern. rial is presented. We have identified zomeric enzymes Mspl and Hpoll. and characterised putative expres Methylation-sensitive RFLPs were sion markers of the montled abnor .. #:. envisaged primarily in order to r----,....~~-~-~:::. _. ma lity for several different stages of screen a pool of oil palm cDNA the micropropagation procedure clones for methylation-dependent (callus, embryoids, leafy shoots and polymorphism, while the aim of greenhouse-harvested leaves). MSAP (methylation-sensitive ampli It is now of paramount importance fied polymorphism) investigations to test the reliability of the markers was to generate a large number of on a wider range of genotypes. This relevant markers, exhibiting a diffe will provide a c1earer picture of their rential methylation pattern depen DIFFERENTIAl DISPlAY ANAlYSIS reliability and enable the selection ding on the normal/montled phe- of specifie markers of interest for notype but independent of the pilot scale clonai conformity testing. genetic origin of clones. CLONING AND SEQUENCING OF POTENTIAl MARKER cDNAs The latter might be achieved by a ln parallel, we addressed the ques number of different techniques, tion of a possible link between the including northern hybridisation, VALIDATION OF MARKERS USING ORIGINAL NORMAL observed hypomethylation and immunodetection (see below) or AND ABNORMAl MATERIAl chroma tin rearrangement, as DNA PŒ. The final choice will depend methylation often parallels the com on a number of factors, notably the paction of a genomic domain [49]. nature of the marker gene and the As a continuation of this work, we developmental stage and tissue cho hope that by isolating oil palm rela sen for the testing. This choice of tives of the Arabidopsis tholiono METI marker will also have to integrate DNA-methyltransferase gene, we the real cost of the technique, in will obtain useful information to help comparison with the production explain how the montled abnorma costs of oil palm vitroplants. litY is generated, dysfunctions of ln parallel with the work described genes of this family having been found to be conformity testing. The differential display (ddRT above, the Cirad-CPIlrd group is also currently linked to a number of developmental abnor PCR) technique [50] was used for this purpose. establishing acollection of systematically sequen malities. The latter technique exploits the rapidity of the ced EST (Expressed Sequence Tag) cDNA clones. polymerase chain reaction (PŒ) to compare This work is being carried out as part of a French Ana/ysis of differentia/ gene expression mRNA abundance between two or more RNA government-funded Genopole project. A key samples extracted from the plant material of inter part of our EST project is centred on studying The fact that the montled abnormality involves est. The isolation, validation and characterisation the functioning of the oil palm shoot apical acharacteristic homeotic modification of oil palm of expression markers of the montled abnorma meristem. We thus plan to build up a collection floral architecture implies that the activity of a lity involves a number of stages summarised in representing genes expressed in the oil palm specifie subset of genes has been altered in soma Figure 4. shoot meristem within specifie tissues, at cer clonai variant plants, at least within the flower The differential display stage provides candi tain developmental stages or in response to and fruit tissues. Attempts are made to identify date markers which must be validated and cha environmental conditions of interest. Our aim floral homeotic genes of the "MADS box" trans racterised using a range of different molecular is to assemble an extensive catalogue of oil pa lm cription factor family which might be affected techniques. Initially, northern blotting is used genes in this way, which will be screened either by the chain of events resulting in the montled to confirm differential expression using the same on the basis of their sequence affinities (simila-
OCL VOL. 8 W 4 JUILLET/AOÛT 2001 299 The originality of oil palm lies in its high capa Tableau 1. Su mmory of differential display marker dota for lealy shoot material. city for multiplication from seed, which allows No of potential markers identified by differential display 46 breeders to build up families of full-sib indivi No of individual c10ned cONA fragments obtained 58 duals which enable an effective evaluation of the general and specific combining abilities of No of (ONAs producing differential signais in first stage 01 northern re-testing 13 their parents, if tested in appropriate struc No 01 (ONAs producing stronger northern signal in normal tissue 6 tures. No of cONAs producing stronger northern signal in abnormal tissue 7 No of cONAs producing consistent signais for several genotypes (confirmed to date) 3 Oi/ pa/m breeding strategy and update on mo/ecu/or markers rity to known genes of interest) or by using high ther DNA methylation plays a direct role in throughput macro- or microarray screening to determining the differential expression pattern Further progresses in oil palm breeding are ham examine their expression patterns. of a given gene, since the activity of the gene pered by several factors, notably: promoter carried in an exogenous plasmid-deriv • the duration of a generation for the crop and Protein studies ed ONA will be probably be unaffected by clo the long selection cycles - ten to twelve years nai conformity in this case. Conversely, if the - that have to be set up on vast experimental ln order to improve our understanding of the differential expression pattern is determined by areas; differential gene expression phenomena under signalling elements further upstream in a regu • the limited knowledge of the genetic diver Iying the appearance of the montled abnorma latory cascade, the activity of the bombarded sity and degree of heterozygosity of the mate litYand to develop strategies for the imple promoter would be expected to be influenced rial tested; mentation of simple and inexpensive clonai by tissue conformity. • the complex phenotypic expression of the conformity testing, we are in the process of For our transformation work, we have chosen main quantitative characters which have been initiating protein studies as a complementary to concentrate on the use of embryogenic sus selected, su ch as: approach to the transcriptome-based work pensions [13] as the starting plant material, since - oil production, which depends on relatively described above. Initial work will be aimed at this should simplify the process of regeneration heritable but negatively correlated characters, characterising variations in the abundance of from tissue culture: low size, abundant and very or on characters influenced by the environment; individual polypeptides in relation to clonai homogenous. A typical example of results - tolerance of vascular wilt (Fusorium oxysporum conformity using two dimensional SOS poly obtained is shown in Figure 5. f.sp. eloeidis); a major disease in Africa; acrylamide gel electrophoresis (20 SOS-PAGE). - tolerance to But Rot disease in South-America; Although the latter technique has been practis - vegetative development: the aim being to ed for many years, it originally suffered from Marker assisted breeding reduce vertical growth in order to prolong the problems of reproducibility which have been economic lifespan of the plantations; considerably improved thanks to recent efforts Oil palm breeding programmes are ail based • the impossibility of determining at the nur in this area [51 J. Thus there has emerged the on a major Sh gene determining three variety sery stage the variety of individuals to be plant new field of proteomics which enables global types [1]: dura (homozygous loci Sh'/Sh') with ed, which results in not testing tenera x tenera comparisons to be made between mRNA and thick-shelled fruits, pisifera (homozygous loci individuals whose 25% pisifero individuals are protein accumulation. We will be using this Sh-/Sh-) which is generally sterile female and too numerous, and not planting plots of pure approach in an attempt to identify early pro produce shell-Iess fruits, and tenero (hetero male pisifera chosen for commercial dura x pisi tein markers of normal or variant tissues. One zygous loci Sh'/Sh-) with medium·thickness fera seed production. inherent advantage of a protein-based approach shells. ln short, in addition to the perennial nature and is that it allows faster progression dimensions of the plant, it is our towards an antibody test compared poor knowledge of the oil palm with RNA studies. Figure 5. Cell suspension calibrated at < 1mm showing transient expression of genome that prevents optimum 3SS: gus three days ofter biofistic transformation. 172 ± 38 spots were counted conventional breeding. Genetic engineering studies on SOOmg FW of plated cel/s (10 replicates). We now have a clear idea of the requirements relating to better Transient expression and stable knowledge of the genetic structure transformation of oil palm tissues of populations, checks on the iden has already been demonstrated [52, tity and heterozygosity of progenies, 53]. more effective studies of the gene As a further complementary tic links between selected characters approach to understand the mole and, above ail, the provision of early cular phenomena underlying the selection aids [54-56]. montled abnormality, we are cur rently in the process of preparing The use of oil palm genetic markers promoter: GUS constructions that was initiated in Malaysia and the UK will be initially tested for their acti at the start of the 1990s, for clonai vitYby transient expression analysis identification and genetic mapping using particle bombardment. This [57,58]. It developed prospectively, may provide indications as to whe- 1.... with the appearance of new tech-
300 LA FILlÊRE AUJOURD'HUI DEMAIN niques: genetic diversity studies of E. guineen sely related species E. oleilera where an optimal kers mapped will primarily be co-dominant, sis using RFLP or RAPD markers [59, 60]; RFLP utility of the SSR markers was observed. locus-specifie and easily transferable from one genotyping of E. guineensis and E. oleilera acces Multivariate data analyses showed the ability of population to another (mostly microsatellites sions [56, 61]; search for cD NA markers of genes SSR markers to efficiently reveal the genetic but also genomic RFLP (Restricted Fragment involved in floral development [62,63] or genes diversity structure of the genus Elaeis in accor Length Polymorphism) or cDNA markers, and coding for fatty acid biosynthesis enzymes [64, dance with known geographical origins and EST (Express Sequenced Tag) markers). Highly 65J; search for RAPD markers of somaclonal measured genetic relationships based on pre polymorphie microsatellite markers will make variants [39]; and RAPD detection of Bud Rot vious molecular studies (Figure 6). High levels up the web of the reference genetic map; these tolerance [66]. The Plant Breeding Institute in of allelic variability indicated that E. guineensis markers are effective in genetic diversity studies UK published [58] the first RFLP genetic map of SSRs will be a powerful tool for genetic studies notably in oil palm [75], and highly suitable for oil palm in 1997. Although it is incomplete, with of the Elaeis genus including variety identifi genome mapping as they provide a wide 860 cM mapped and 24 linkage groups com cation and intra- or inter-specifie genetic map genome coverage [76]. These markers will be pared to 16 chromosome pairs, the map was a ping (Table 2). PCR amplification tests, from a topped up with RFLP and cDNA "anchor first step towards a rational use of molecular subset of 16 other palm species, and allele points". Finally, AFLP (Amplified Fragment markers. sequence data showed that E. guineensis SSRs Length Polymorphism) markers will be used to Furthermore, molecular analysis of E. a/eilera germ are putative transferable markers across palm saturate the reference linkage map. plasm has been realised using cDNA and geno taxa. In addition, phenetic information based mie RFLP probes [67] and the joint use of selec on SSR flanking region sequences makes E. gui Establishment 01 a mufti-parent consensus map tion indexes and molecular markers was employed neensis SSR markers a potentially useful mole The theoretical work performed by Muranti [77, to optimise the breeding scheme [68, 69]. cular resource for any researcher studying the 78] showed that a multi-parent consensus map, phylogeny of palm taxa. established with several parents (at least four to Marker-assisted breeding strategy six) and full-sib families linked within a diallel for ail palm Establishing a reference genetic map or factorial design is the most effective in the ln addition to providing further knowledge of search for Qn suitable for use in marker-assist Biomolecular research applied to marker-assist the genome, the aim of drawing up reference ed breeding. In addition to ensuring more accu ed oil palm breeding is a long-term plan. The maps is a rational choice of markers and parents rate detection compared to a single two-parent different stages identified for oil palm [70] are to be used la ter in the plan. Acontrol dura Deli map, it also enables an evaluation of the effects detailed in this chapter. x tenera La Mé F1 progeny widely used in the of QTL depending on their type (additive, domi breeding scheme was chosen (cross DA1OD x nance) and on different genetic backgrounds. Mass development al PCR based-markers in ail LM2T). The homologous and heterologous mar- Such a multi-parent map will be established by palm Simple Sequence Repeats (SSRs), also called microsatellites, are tandem arrays of simple Figure 6. Factorial anolysis 01 correspondences perlormed on 20 single-locus mierosatellile markm aver 18 acces sions 01 E. guineensis (Africa), 19 accessions al E. oleifera (Brozil, Central America, French Guyana, Peru and nucleotide motifs that are ubiquitous compo Surinam) and 1accession of B. adora. Note: Axis 1and 2 represent 20.2% of the total molecular variability. nents of eucaryotic genomes [71]. Inherited in a Mendelian fashion [72] their hypervariable length polymorphism is simply revealed by the French Guiana Polymerase Chain Reaction (PCR) using flan Surinam king primers that generate co-dominant mar 0 kers. Development of oil palm microsatellite markers has been undertaken since 1998, with the view to developing genetic diversity stu Elaeis guineensis dies, to variety identification, to pedigree ana !\frica Iysis as weil as to genome mapping and QTL Elaeis oleifera detection towards marker-assisted selection activities. A quick and simple technique for building micro Axis 1 (11.2%) satellite-enriched libraries was developed [73J, from a hybridisation-based capture methodo ____ 0 ® Central America logy using biotin-Iabelled microsatellite oligo probes and streptavidin-coated magnetic beads. About 200 functional SSR primer pairs have G Peru already been developed for oil palm from micro satellite clones which have been sequenced in collaboration with the French National Sequencing Centre in Evry France (Genoscope). The characterisation of 21 SSR loci was publis hed [74] together with primer sequences, esti Barcella adora 0 mates of allele size range as weil as expected heterozygosity in E. guineensis and in the clo-
OG VOL. 8 NO 4 JUILLET/AOÛT 2001 301 . ....
Tableau 2. Type 01 repeats and average values 01 SSR al/ele numbers, expeeted heterozygosity (He) and probability 01 identity (PI) 0120 E. guineensis SSR loci in E. gui neensis and E. oleifera.
Sample SSR Repeats # AIIeles # Alleles # Shared Total # He PI motif Number E.g. Lo. aile les aile les Lg. Lo. Lg. + E.o. E.g. E.o. 9 (GA)n 17 7.1 7.3 3.0 11.4 0.7 0.7 0.8 0.02-0.15 0.02-0.38 7 (GT)n 10 4.3 3.9 1.6 6.6 0.4 0.4 0.6 0.02-1.00 0.09-1.00 4 (CCG)n 6 2.7 3.5 1.7 4.5 0.4 0.6 0.6 0.11-0.40 0.12-0.23 Average - - 5.2 5.3 2.2 8.4 0.5 0.6 0.7 0.3 0.2 Polymorphie markers 80% 95% 100%
Cirad in collaboration with PT SOCFIN A F1-type progeny (dura x tenera) of 90 indivi approach. The AFLP·BSA marker and the gene Indonesia, using microsatellite and cDNA mar duals obtained from a DA115D x LM2T cross tic map of LM2T are an important step towards kw on an existing factorial genetic design was chosen to carry out genetic mapping of the marker-assisted selection of the 5h gene, and already observed for most vegetative and pro oil palm and its Sh gene, in the LM2T parent. of other genes of agronomie interest in oil palm. duction characters. The map population consists The genetic map of LM2T, constructed with of about 600 palms and several dura x tenera MAPMAKER at LOD score =5.0 and r = 0.3 Usefu/ applications of genetic mapping full-sib families, including the reference map (apart from minor exceptions), distributed aset • The molecular dissection of the chromoso population, obtained from different La Mé, Deli of 149 AFLP or microsatellite markers in 15 lin mal region surrounding major gene Sh, coding and Yangambi parents. Ali individual maps of kage groups, 3 pairs and 6 unlinked markers for the existence or lack of a shell, with a view the system will be connected between them (Figure 8). The number of linkage groups, close to c10ning and (intragenic) labelling of the relat selves by common parents and by co-segregat to the number n = 16 pairs of chromosomes of ed genes responsible for fruit morphology and ing microsatellite or eDNA/EST markers. the plant, and a total map size of 1,355 cM fertility (degree of sheillignification, kernel already indicate relatively good coverage of the volume in dura and tenera varieties, female ste Search for worthwhi/e QTL or major genes genome. The AFLP-BSA marker, which had rility of pisifera genotypes). QTL for vegetative and yield characters, as weil revealed a co-dominance reading of the Sh+ • The characterisation, c10ning and tagging of as of vascular wilt tolerance are detected by stu gene on the dura and tenera phenotypes, was vascular wilt tolerance genes. dying the correlation existing between the mar likewise mapped in LM2T. The gene 5h and its • Molecular studies of genetic diversity in oil palm, kers and the phenotypic characters of the indi AFLP-BSA marker were mapped onto the long which will be based partly on QTL markers and viduals chosen for the individual or consensus est linkage group of the map (219.5 cM), at will determine the potential for exploiting natu maps. The variability of QTL effects due to the 7.2 cM or 12.6 cM according to the respective rai diversity, particularly to predict heterosis. environ ment will be assessed on a control pro JOINMAP or MAPMAKER genetic mapping soft • The search for plant material tolerant to Bud geny duplicated under different ecological ware. Rot in Latin America, through the genetic map conditions. These results demonstrated the pertinence of ping of tolerance genes in the American spe At the present time, two complementary BSA analysis in the search for molecular mar cies E. oleifera [81] with a view to their intro approaches have been followed in the aim of kers of the Sh gene, and the paramount inter gression into E. guineensis by marker-assisted identifying AFLP or microsatellite markers link est of genetic mapping as a complementary backcrossing. This latter project is based on the ed to the Sh gene, governing shell genetic mapping of an inter-speci thickness: i) by BSA (Bulk Segregant fie back-cross of first generation Analysis) of segregant groups [79], Figure 7. Candidate AFLP marker 01 the Sh+ ol/ele evidenced by bulk segreganl (BC1) for which the resistance to an efficient methodology for ana/ysis 01 groups issued tram the se/ling 01 a tenera LM2T parent. Note: D = dura Bud Rot of each genotype will be DNA bu/k; T=tenera DNA bulk; P=pisifera DNA bulk. detecting major genes and ii) by evaluated through the field test of genetic mapping [80]. A total of clonai descendants planted in an 124 EcoRI/Msel AFLP primer pairs area strongly affected by the disease. and 88 microsatellite primer pairs was used to analyse 3 dura, tenera Validation of mo/ecular markers and pisifera segregant groups, each Field Trial Systems (FTS) consisting consisting of the DNA of 8 indivi of several populations to be used for duals obtained by selfing of a tenera future marker·assisted breeding pro ...... ---Candidate AFLP-B5A parent LM2T. Out of a total of grammes will be established using marker AggCAA2ü 9,3 30 loci screened by the AFLP tech new factorial genetic designs. Trials (132 bp) nique, an AFLP-BSA candidate marker will be planted at different locations AggCAA20 was identified (Figure 7) consisting of 5,000 genotypes and then validated by individual analyses p involving at least 20 parents (full-sib of the DNA making up the mixes. families). Microsatellite screening did not reveal The objectives are to test the accuracy any marker on these mixes. of molecular markers on multi-parent
302 LA FILlËRE AUJOURD'HUI DEMAIN Figure 8. AFLP and microsalellite ail palm linkage map 01 the lenero LM2T parent, built with the MAPMAKER 3.0 software (LOD score =5.0; r =0.3). Note: the Sh gene and ils AFLP·BSA marker are mapped on the linkage group number 1.
2 .5 3 4 5 6 7 AI\gC\,\) \ ,\({{'\T{ll) NCCTI04r "V::CG\TU7r Sh } 1 '~X't~J~\!ll ·1.,] loCi 7..1 mr\;(lJ..\25 121> }53 nlf.g0775 IIU '~:~[{~~~g~ lK. {- \~( ICOI. ](dJ AggCAA20r "\(AY\H~!,r 'JI U 14.') ;\«(\;;(1\r l11[gOl 'i7 151 "1} ml,
LOD J.5 A.·\CCTgllr nu {\ggÜ\r\(J6<
17] 1Local aggregation oÎ rnarkers at the LOD score indicated .\rçc ,\0 rnE!,>111lJ2r ,9 201 A/\gG-\A241
mF K0801r ll.n 1(\ \ LOO-J.U '\/\g<'A;\il1j !\r\{~G\T02 lb.lJ
m~g07t)ij H.U :\,\CCTTI(Jr
9 10 11 12 13 14 ;\AgeA/\l!! NAnglO ;~R~P~:~i~~ 'dl \.7 2I i\(J\~~'()7( )(L2 1.1 '\g(CTC1B 4,~ :'~(\~~~~J\tm~ l _ i\ fl(TCl2 '- 2(J,\{>Cc co tM'<~"~<,«'" 2\R{ l/,h yy~~t,\~i1 Wb ,....J\CCT~05 ,.IAAC,,.IUi A"'CCATI \ :\;!,gLî(()hr LODU 11,1) 275 '\~,~Ai6 mEgOl9S Udl 12.7 ~\g~(At\2b 17.5 AM Il1fgl772r 15 16 17 18 mEgOI77 !1lE.g0210 ,lgCCAg02 2,2----.c::c::~-{{lllr B.H ltl.1 9.1 -{[ MUjlgOJ .. 1j±; mEgU'î21! m[g0772r ]j,a mEg()77 \ (j(' ~ nlFt'07t11r . ,- AglCTCl1 and connected full-sib families issued from exten assisted detection and fast introgression of efficiency of conventional breeding in terms of ded genetic backgrounds and dedicated to awider important Elaeis oleifera characters into oil palm: accuracy and time saving. lt is an early selec identification of intra- and inter-population QTL low height increment and high oil quality, but tion tool. It will be easier to determine the geno diversities. Also, the project results will integrate also genetic resistance factors to the lethal type of material to be tested at an early stage, into current oil palm breeding programmes in the diseases Ganoderma, Fusarium wilt and Bud Rot. rather than measuring certain phenotypic cha form of an applied multi-characters marker-assist racters at each generation, for ten years or more. ed breeding strategy for worthwhile genes. Various Perspectives in marker-assisted This will limit the time lapse between selected parents will be used including palms suspected or breeding of ail palm generations, fasten genetic gain and so increase known to present genetic resistance factors to the the expected quality of commercial seed. lethal diseases caused by Ganoderma or Fusarium Molecular markers data will be associated with Major applications will concern the following 0xyspolllm. selected phenotypic characters to define and areas: Some trials will test inter-specific back-crosses apply the marker-assisted breeding scheme. • variability management (germplasm, combi of first or second generation for the marker- Such use of molecular markers will increase the ning ability groups), OCL VOL. 8 N° 4 JUILLET/AOÛT 2001 303 • .... • genotyping for characters highly influenced a means of establishing an early clonai confor 3. JACQUEMARD JC, BAUDOUIN L, NOIRET lM by the environ ment or costly to measure, mity testing procedure. A number of genes (1997). Le Palmier à huile. In : CHARRIER A, JAC • control and monitoring of recombinations, whose expression appears to be modulated in QUOT M, HAMON S, NICOLAS D, eds. • predicting genotypic values for complex cha tissue cultures according to their mantled sta L'amélioration des plantes tropicales. Paris: Édi• tions Repères, Orstom/Cirad, 507-32. racters, tus have been identified and characterised. 4. NOIRET JM (1981). Application de la culture in • predicting the value of a cross from informa Efforts will now be directed towards the deve vitro à l'amélioration et à la production de maté tion about the parents. lopment of early clonai conformity tests. Markers riel clonai chez le palmier à huile. Oléagineux, 36 : Genotype identification and legitimacy analyses will be tested on a wide range of genotypes to 123-6. (determination of paternity, reproduction assess their reliability and studies will be per 5. HARTLEY CWS (1988). The Oil Palm. Tropical schemes) are one major application. In oil palm, formed to identify when in the micropropaga Agriculture Series. London: Longman, 761 p. molecular markers will be used to select dura, tion procedure testing should best be carried 6. KRIKORIAN AD (1989). The context and strate tenera or pisifera individuals at the nursery stage. out. It is important to assess what kind of metho gies for tissue culture of date, african oil and eoeo ln the same way, it will be possible to detect dology is the most appropriate for clonai confor nut palms. In: VIBHA DHAWAN, ed. Applications and then plant pisifera individuals in male parent mity testing by comparing RNA, protein and of bioteehnology in Forestry and Horticulture. New plots for tenera seed production. It will be partly DNA (PCR) based approaches. York: Plenum Press, "9-44. 7. MEUNIER J(1975). Le palmier à huile américain possible, using gene markers, to evaluate the Molecular markers data will be associated with Elaeis melanoeoeca. Oléagineux, 30: 51 -61. genetic value of these pisifera individuals, which selected phenotypic characters to define and 8. RIVAL A(2000). Soma tic embryogenesis in oil was impossible in the field until now due to their apply the marker-assisted breeding scheme. palm. In: Jain SM, GUPTA PK, NEWTON RI, eds. female sterility. Lastly, effective management of Such use of molecular markers will increase the Somatie embryogenesis in woody plants, vol. 6, the entire variability of the E. guineensis species efficiency of conventional breeding in terms of chap. 8. Dordrecht (The Netherlands): Kluwer will be a possibility, with field testing of tenera accuracy and time saving, Academic Publishers, 249-90. x tenera progenies, of which only tenera indi 9. SMITH WK, 10NES LH (1970). Plant propagation viduals will be retained. through cell culture. Chem & Ind, 44: 1399-401. Automatic genotyping will concern the main Acknowledgements 10. CORLEY RHV, BARRETT IN, 10NES LH (1977). worthwhile parents and the survey populations Research work described in this chapter was Vegetative propagation of oil palm via tissue cul· will be integrated into the oil palm breeding conducted under a joint research programme ture. Oil Palm News, 22: 2-8. scheme. Here, biomolecular results, in terms of between IRD (ex-Orstom; Institut de recherche ". RABECHAULT H, AHÉE 1, GUÉNIN G (1970). Colonies cellulaires et formes d'embryoïdes obte favourable gene detection and evaluation with pour le développement) and Cirad-CP (Centre nues in vitro à partir de cultures d'embryons de molecular tools, will be applied to the whole de coopération internationale en recherche agro palmiers à huile (Elaeis guineensis laeq. var. dura breeding scheme. nomique pour le développement - Département Bece). CRAS Série D, 270: 3067·70. cultures pérennes). 12. REDENBAUGH K(1993). Synseeds: applications of Research on soma clonai variation was partially synthetie seeds to erop improvement. London: CRC Conclusion funded with the aid of a grant from the Press, 3-7. Malaysian Palm ail Board (formerly PORIM) 13. TOUCHET (de) B, DUVAL Y, PANNETIER C(1991). Biotechnological approaches applied to oil palm through aCollaborative Research Project (nOCRB Plant regeneration from embryogenic suspension breeding have considerably increased in their 96-001 ). culture of oil palm (Elaeis guineensis Jacq). Plant importance, not only in micropropagation, but The authors thank colleagues involved in the Cell Reports, 10: 529-32. also in marker-assisted breeding, and more ail Palm Project: Jean-Luc Verdeil at IRD 14. TEIXEIRA JB, SONDHAL NR, NAKAMURA T, KIRBY EG (1995). Establishment of oil palm cell sus generally, in physiological and molecular stu GeneTrop; Tristan Durand-Gasselin, Philippe pensions and plant regeneration. Plant Cell, Tissue dies of the expression of genes of paramount Amblard and Françoise Potier at Cirad-CP. agronomie value (flowering, abscision, disease and Organ Culture, 40: 105·11. Biotechnology research would not have been 15. DUVAL Y, ENGELMANN F, DURAND-GASSELIN T resistance, etc). possible without the excellent collaboration of (1995). Somatic embryogenesis in oil palm (Elaeis Recent results in micropropagation using our colleagues working in palm oil producing guineensis Jacq.). In: BAJAJ YPS, ed. Somalie embryogenic suspension open the way for the countries : CNRA at La Mé in Côte d'Ivoire, embryogenesis and Synthetie seed l, Bioteehnology use of strategies based on the "artificial seeds" INRAB at Pobé in Benin, SOCFINDO and IOPRI in Agriculture and Forestry. Berlin: Springer Verlag, concept for oil palm. in Indonesia, and FELDA and MPOB in Malaysia. vol. 30: 335-52. Studies on global methylation rates provided a 16. 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