Occurrence of aromatic cytokinins in oil palm (Elaeis guineensis Jacq.)

The natural occurrence of 6-benzylaminopurine, 6-(2-hydroxybenzylamino)purine (ortho-topolin), 6-(3-hydroxybenzylamino)purine (meta-topolin), their ribosides and 9-glucosides is reported using specific antibodies to these groups of compounds in high performance liquid chromatography/enzyme-linked immunosorbent assay (HPLC/ELISA). Compounds were identified by their retention times and differential cross-reactivities with six antisera in analyses carried out in two laboratories using different HPLC gradient systems. Identities were confirmed by immunoaffinity purification followed by HPLC with on-line UV spectrum analysis. Further confirmation of the occurrence of ortho-topolin riboside and isopentenyladenine-9-glucoside was obtained from gas chromatography-mass spectrometry analysis of permethylated HPLC fractions of an extract of oil palm tissues. The aromatic cytokinins, and in particular, ortho-topolin riboside, were found in a variety of oil palm tissues at concentrations exceeding those of the isoprenoid cytokinins, zeatin, isopentenyladenine, dihydrozeatin, and their ribosides. The 9-glucosides of isopentenyladenine and zeatin were more abundant than those of the aromatic types. The cross-reactivity of benzyladenine compounds with antibodies to isopentenyladosine is discussed in relation to the interpretation of ELISA data.Abbreviations BA N ⁶-benzyladenine - HPLC high performance liquid chromatography - ELISA enzyme-linked immunosorbent assay - GC-MS gas-chromatography-mass spectrometry - mT meta-topolin - oT ortho-topolin - TEAA triethylammonium acetate - IAC immunoaffinity chromatography - SPD spectral photodiode     

Plant regeneration from embryogenic suspension cultures of oil palm (Elaeis guineensis Jacq.)

Suspension cultures of oil palm (Elaeis guineensis Jacq.) were established in a medium with 80 or 100 mg.l^–1 2,4-dichlorophenoxyacetic acid and 1 g.l^–1 activated charcoal, from calli producing embryogenic cells and protruding proembryos. The suspension was composed of meristematic clumps, breaking away and giving rise to new smaller aggregates. Under the best conditions, the initial weight increased about 4 fold in one month. Embryo differentiation was achieved when plating the clumps either directly, or after a phase in hormone-free liquid medium. In the second case, early maturation occurred in liquid medium. Secondary embryogenesis was reduced, and up to 18.1 % embryos formed shoots. Rooted plants were sucessfully transferred to soil.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Assaying lipase activity from oil palm fruit (Elaeis guineensis Jacq.) mesocarp.

The mesocarp of mature oil palm fruit undergoes intensive triglycerides hydrolysis upon abscission and bruising. This generates such a high amount of free fatty acids that the oil might become unfit for human consumption without appropriate refining. The lipase (EC 3.1.1.3) involved in the breakdown of the oil is not stable after homogenization of the tissue in aqueous buffers. In this study, we have devised a solvent-based procedure that allowed us to obtain fractions with stable lipase activity. Using these fractions, we have determined the optimal conditions for assaying mesocarp lipase activity. The activity was highest at a temperature of 35 degrees C and a pH of 9. The lipase was found to be strictly calcium dependent. The specific activity of the lipase measured in optimal conditions was found to be 33 mumol fatty acids released min(-1) mg(-1) protein using olive oil as substrate. The mesocarp contains about 190 U of lipase g(-1) fresh weight. This activity was found to be inhibited by the lipase inhibitor tetrahydrolipstatin (THL), suggesting that the lipase is a serine hydrolase.     

Quantitative trait loci for yield components in oil palm (Elaeis guineensis Jacq.)

The development of an oil palm RFLP marker map has enabled marker-based QTL mapping studies to be undertaken. Information from 153 RFLP markers was used in combination with phenotypic data from an F₂ population to estimate the position and effects of quantitative trait loci (QTLs) for traits including yield of fruit and its components and measures of vegetative growth. The mapping population consisted of 84 palms segregating for the major gene influencing shell thickness. Marker data were analysed to produce a linkage map consisting of 22 linkage groups. The QTL mapping analysis was carried out by interval mapping and single-marker analysis for the unlinked markers; significance thresholds were generated by permutation. Using both single-marker and interval-mapping analysis significant marker associated QTL effects were found for 11 of the 13 traits analysed. The results of interval-mapping analysis of fruit weight, petiole cross section and rachis length, and ratios of shell:fruit, mesocarp:fruit and kernel:fruit indicated significant (P<0.05) QTLs at the genome-wide threshold. The putative QTLs were associated with between 8.2% and 44.0% of the phenotypic variation, with an average of 27% for the single-marker analysis and 19% for the interval-mapping analysis. The higher percentage of phenotypic variation explained in the single-marker analysis, when compared to the interval-mapping analysis, is likely to be due to the lower stringency associated with the single-marker analysis. Large dominance deviations were associated with a sizeable proportion of the putative QTLs. The ultimate objective of mapping QTLs in commercial populations is to utilise novel breeding strategies such as marker-assisted selection (MAS). The potential impact of MAS in oil palm breeding programmes is discussed. Received: 26 June 2000 / Accepted: 24 October 2000     

QTL detection by multi-parent linkage mapping in oil palm (Elaeis guineensis Jacq.)

A quantitative trait locus (QTL) analysis designed for a multi-parent population was carried out and tested in oil palm (Elaeis guineensis Jacq.), which is a diploid cross-fertilising perennial species. A new extension of the MCQTL package was especially designed for crosses between heterozygous parents. The algorithm, which is now available for any allogamous species, was used to perform and compare two types of QTL search for small size families, within-family analysis and across-family analysis, using data from a 2 × 2 complete factorial mating experiment involving four parents from three selected gene pools. A consensus genetic map of the factorial design was produced using 251 microsatellite loci, the locus of the Sh major gene controlling fruit shell presence, and an AFLP marker of that gene. A set of 76 QTLs involved in 24 quantitative phenotypic traits was identified. A comparison of the QTL detection results showed that the across-family analysis proved to be efficient due to the interconnected families, but the family size issue is just partially solved. The identification of QTL markers for small progeny numbers and for marker-assisted selection strategies is discussed.     

Application of X-ray microanalysis to cell suspensions of oil palm (Elaeis guineensis Jacq.)

X-ray microanalysis has been used to determine the elemental composition of oil-palm (Elaeis guineesis) cell suspensions without the use of cryoprotectants. Results based on individual cells were gathered over a typical growth cycle of 14 d. During the log phase (5–7 d) there is an increase in the number of cells containing high concentrations of both K (400 mmol kg^-1 dry weight) and P (400 mmol kg^-1 dry weight). Morphologically these cells had thin cell walls and were frequently joined to other cells (two to five cells per clump).     

Cryopreservation of oil palm (Elaeis guineensis Jacq.) somatic embryos involving a desiccation step

Summary The standard cryopreservation process previously developed for oil palm clones using shiny white, finger-like somatic embryos could be applied in some cases to standard cultures. Its efficiency was markedly improved by completing the 7-day pregrowth period on 0.75 M sucrose by an additional dehydration period carried out either by placing the embryos in the air current of the laminar flow cabinet or in an air tight box containing silica gel. This improved process was successfully applied to 7 different clones. It will facilitate the routine uof cryopreservation for oil palm cultures.     

Somaclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis

Elaeis guineensis Jacq.) currently hampers the scaling-up of clonal plant production. In order to investigate the relationship between the “mantled” somaclonal variant and possible alterations in genomic DNA methylation rate, two complementary approaches have been used. HPLC quantification of relative amounts of 5-methyl-deoxycytidine has shown that global methylation in leaf DNA of abnormal regenerants is 0.5–2.5% lower than in their normal counterparts (20.8% vs 22%, respectively). When comparing nodular compact calli and fast growing calli, yielding respectively 5% and 100% of “mantled” plantlets, this decrease was up to 4.5% (from 23.2 to 18.7%). An alternative method, the SssI-methylase accepting assay, based on the enzymatic saturation of CG sites with methyl groups, gave convergent results. This work demonstrates that a correlation exists between DNA hypomethylation and the “mantled” somaclonal variation in oil palm. Received: 9 July 1999 / Revision received: 15 October 1999 / Accepted: 26 October     

RAPD linkage mapping of the shell thickness locus in oil palm (Elaeis guineensis Jacq.)

Shell thickness is an important trait in oil palm breeding programs and is the basis for the classification of the varieties of oil palm into the types dura, tenera and pisifera. This trait seems to be controlled by a single locus, with two alleles (sh ⁺ and sh ⁻) showing codominant expression. Two single-tree linkage maps were constructed for a maternal tenera (sh ⁺ sh ⁻) palm and for a paternal pisifera (sh ⁻ sh ⁻) palm using the pseudo-testcross mapping strategy in combination with RAPD markers through the analysis of an F₁ tenera×pisifera progeny. A total of 308 arbitrary primers were screened in a sample of eight F₁ plants and 121 markers were detected in a testcross configuration. An average of 1.66 polymorphic marker per selected primer were identified in this cross. At LOD 5.0 (with some few exceptions) and θ=0.25 the maternal tenera map included a total of 48 markers distributed in 12 linkage groups or pairs of markers (449.3 cM) while the paternal pisifera map included 42 markers distributed in 15 linkage groups or pairs of markers (399.7 cM). We used RAPD and bulked segregant analysis (BSA) to identify markers more tightly linked to the sh ⁺ locus. A total of 174 new primers not previously used in the linkage analysis were screened using bulks of DNA extracted from plants selected for the contrasting shell-thickness phenotypes. Two RAPD markers (R11–1282 and T19–1046) were identified to be linked on both sides of the sh ⁺ locus on linkage group 4. The estimated map distances from sh ⁺ to R11–1282 and to T19–1046 were 17.5 cM and 23.9 cM, respectively. The results demonstrate the usefulness of RAPD markers and the pseudo-testcross mapping strategy for developing genetic linkage information, and constitute an important step towards early marker-assisted selection for shell thickness in oil palm. Received: 21 February 1999 / Accepted: 29 April 1999     

Ecological studies on Pythium splendens braun in oil palm (Elaeis guineensis Jacq.) plantation soils

Summary The isolation of Pythium splendens Braun by a root-baiting technique showed that the infective propagules of the fungus were numerically 12 times as great in cultivated soil as in non-cultivated secondary forest soil.In cultivated oil palm nursery soil, the propagules of the fungus were found to be most abundant at a depth of between 15 and 30 cm below ground level. The recovery of the fungus from the soil around the roots of diseased oil palm seedlings was 4 times as great as that from around the roots of healthy seedlings. Farmyard manure increased the recovery of the fungus from naturally infested soil, whilst green manure reduced it.The recovery of the fungus from soil declined sharply with decrease in soil moisture content and also with the length of storage of air-dried soil.The recovery of the fungus from nursery soil was high in July–October and low in November–January and these variations corresponded with seasonal fluctuations in soil moisture content.     

Evaluation of five promoters for use in transformation of oil palm (Elaeis guineensis Jacq.)

The efficiency of GUS (-Glucuronidase) gene expression in embryogenic callus and young leaflets of mature and seedling palm after microprojectile bombardment with five constructs (pEmuGN, pAHC25, pAct1-F4, pGH24 and pBARGUS) was evaluated to identify the most suitable promoter(s) to use in transformation attempts in oil palm. Expression of the GUS gene driven by theEmu, Ubi1, Act1 35S orAdh1 was assayed, both histochemically and fluorometrically, from a total of 200 plates of tissues in eight independent experiments two days after bombardment. A completely randomized experimental design was used for each experiment, and the data analysed by ANOVA and Duncan's Multiple Range Test. The expression level of GUS driven by theEmu orUbi1 promoters was significantly higher than that of the Act], 35S and Adhl promoters in many experiments, and that of theAdhl was significantly lower than those of the other four promoters. Both histochemical and fluorometric data indicate that in embryogenic callus, the expression of theEmu promoter was higher than that of theUbi1 whereas in young leaflets from mature palm the Ubi1 expression was stronger. The performances of the five promoters were also tested in tobacco callus using a fluorometric GUS assay. The activity of the 35S promoter was highest, and significantly different from that of all the other promoters except theEmu, and that of theAct1 promoter was lowest. These results indicate that either theUbil orEmu promoter should facilitate the expression of desired genes in oil palm and aid in development of an efficient stable transformation system.Abbreviations GUS -Glucuronidase - EC embryogenic callus - YLMP young leaflet from mature palm - YLSP young leaflet from seedling palm - MU methyl umbelliferone - MUG 4-methyl--D-glucuronide - X-glue 5-bromo-4-chloro-3-indoyl-glucuronide - Ubil maize ubiquitin 1 - Actl rice actin 1 - Adh1 maize alcohol dehydrogenase 1 - Emu a recombinant truncated maize alcohol dehydrogenase 1 - ANOVA Analysis of variance - DMRT Duncan's Multiple Range Test Communicated by W A. Parrott     

Microsatellite-based high density linkage map in oil palm (Elaeis guineensis Jacq.).

A microsatellite-based high-density linkage map for oil palm (Elaeis guinensis Jacq.) was constructed from a cross between two heterozygous parents, a tenera palm from the La Mé population (LM2T) and a dura palm from the Deli population (DA10D). A set of 390 simple sequence repeat (SSR) markers was developed in oil palm from microsatellite-enriched libraries and evaluated for polymorphism along with 21 coconut SSRs. A dense and genome-wide microsatellite framework as well as saturating amplified fragments length polymorphisms (AFLPs) allowed the construction of a linkage map consisting of 255 microsatellites, 688 AFLPs and the locus of the Sh gene, which controls the presence or absence of a shell in the oil palm fruit. An AFLP marker E-Agg/M-CAA132 was mapped at 4.7 cM from the Sh locus. The 944 genetic markers were distributed on 16 linkage groups (LGs) and covered 1,743 cM. Our linkage map is the first in oil palm to have 16 independent linkage groups corresponding to the plants 16 homologous chromosome pairs. It is also the only high-density linkage map with as many microsatellite markers in an Arecaceae species and represents an important step towards quantitative trait loci analysis and physical mapping in the E. guineensis species.     

Variation in oil palm (Elaeis guineensis Jacq.) tissue culture-derived regenerants revealed by AFLPs with methylation-sensitive enzymes

Tissue culture-derived plants of oil palm (Elaeis guineensis Jacq.) can develop abnormal flowers in which stamen primordia are converted into carpel-like tissues (mantled fruit). This abnormality can be heritable; individual palms may show variation in mantling and reversion to the normal phenotype over time has been observed. Four sets of ortets (mother plant used as tissue source) and ramets (regenerated plants) were compared using standard amplified fragment length polymorphism (AFLP) analysis and AFLPs using methylation-sensitive enzymes. No polymorphisms were found when standard AFLPs were produced with ten different primer combinations. In contrast, when methylation-sensitive AFLPs were used, polymorphisms were detectable. Polymorphisms appeared as new bands in the ramets, suggesting that a reduction in methylation had occurred during tissue culture. The highest number of polymorphic bands (0.3%) was obtained when HpaII was used as the restriction endonuclease, indicating that the loss of methylation had occurred most frequently at the internal C within the HpaII recognition sequence 5’-CCGG-3’. Conversion of nine of the polymorphic bands into probes for Southern analysis confirmed that these were not due to partial digestion of the AFLP templates and showed that the majority were single-copy sequences. The exceptions were fragments showing homology to 25S ribosomal RNA genes and the chalcone synthase gene family. Examination of the Southern blots suggested that most of the single-copy sequences were partially de-methylated, and one example was found in which de-methylation affected only one allele. No polymorphism was consistently different between normal and abnormal clones in all the sets. This suggests that, whilst this method is an effective way of detecting variation in tissue culture-derived plants, different approaches will be required to identify the causal basis of the mantled fruit abnormality. Received: 25 May 2000 / Accepted: 28 August 2000     

Differential responses to somatic embryogenesis of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.)

To assess the potential of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.) to somatic embryogenesis and somatic embryo proliferation, mature zygotic embryos of nine commercial genotypes of E. guineensis (BRSC2001, BRSC2328, BRSC2301, BRSC3701, BRSCM1115, BRSC7201, BRSC2528, BRSC2501, and BRSCN1637) were used. Explants were incubated on Murashige and Skoog (MS) supplemented with 450 μM picloram, 3.0 % sucrose, 500 mg l^?1 glutamine, and 2.5 g l^?1 activated charcoal, and gelled with 2.5 g l^?1 Phytagel. After induction, for differentiation and maturation, the embryogenic calli (ECs) were transferred into fresh medium supplemented with 0.6 μM naphthaleneacetic acid (NAA) and 12.30 μM 2-isopentenyladenine (2iP) or 40 μM picloram in combination with 0.3 g l^?1 activated charcoal, and 500 mg l^?1 glutamine. Somatic embryos were converted into plants on MS medium with macro- and micro-nutrients at half strength, 2 % sucrose, and 2.5 g l^?1 activated charcoal, and gelled with 2.5 g l^?1 Phytagel. In general, zygotic embryos swelled after 14 days. Primary calli, which were observed in all the genotypes after 45–60 days of culture, eventually progressed to ECs at 90 days. At this time, scanning electron microscopy (SEM) analysis showed cellular differences between compact and friable calli. After 150 days in the induction phase, the ECs with proembryos that were transferred to the medium for differentiation and maturation, differentiated asynchronically into somatic embryos at globular and torpedo stages. The results showed that BRSC2328 and BRSCM1115 had the highest potential for EC formation (90–100 %) and somatic embryo differentiation (40.7 and 52.5 somatic embryos per callus, respectively) when compared to other genotypes. After approximately 90 days of culture on MS basal medium without growth regulators, protrusion of the leaf primordia was observed, characterizing the onset of germination of the somatic embryos into plants.     

Assessment of nuclear,mitochondrial and chloroplast RFLP markers in oil palm (Elaeis guineensis Jacq.)

A variety of DNA probes was used to screen a diverse set of oil palm accessions in order to identify markers with a utility in genotype discrimination. This survey included samples of the commercial oil palm native to Africa (Elaeis guineensis Jacq.), the closely-related South American species [E.oleifera (HBK) Cortes] and inter-specific hybrids of the two. Of 106 major chloroplast bands none showed differences between E. guineensis and E. Oleifera. Mitochondrial and ribosomal probes were more informative inter-specifically (the former allowing identification of the maternal inheritance of mitochondria) and may be useful in hybrid breeding programmes; however, they were unable to identify polymorphism within E. guineensis. In contrast, low-copy nuclear genomic clones were able to identify intra-specific variation, though in most cases they revealed a relatively small number of allelic variants. One DNA probe showed a much larger number of band variants, revealing ten patterns amongst 13 E. guineensis accessions, and should prove useful in genetic fingerprinting and evaluation of oil-palm germplasm collections.     

Vibrating probe measurement of ionic currents around developing embryo of oil palm (Elaeis guineensis Jacq.)

The current patterns around zygotic embryos of the oil palmembryo (Elaeis guineensis Jacq.) were investigated. Ionic currentsaround the embryo were measured at different stages in its developmentusing a two-dimensional vibrating probe system. The averagecurrent density was found to be in the order of 1–µAcm^–2. In the early developmental stage, the current wasfound entering the cotyledon and leaving the radicle. Both themagnitude and direction of this current changed when embryosat different developmental stages were observed. The currentswere found to enter the region in the process of differentiationor elongation, and in some cases this could be the reverse ofthe current direction in the preceding stage. Within one ofthe stages of the embryos, current density was correlated withthe rate of elongation. A very large inward current (up to 20µAcm^–2) was also detected at the point where the shoot wasbreaking through the surface. This was probably caused by theso-called ‘injury current’. Key words: Ionic currents, vibrating probe, oil palm embryo, E. guineensis, injury current     

Natural 13C distribution in oil palm (Elaeis guineensis Jacq.) and consequences for allocation pattern

Oil palm has now become one of the most important crops, palm oil representing nearly 25% of global plant oil consumption. Many studies have thus addressed oil palm ecophysiology and photosynthesis‐based models of carbon allocation have been used. However, there is a lack of experimental data on carbon fixation and redistribution within palm trees, and important C‐sinks have not been fully characterized yet. Here, we carried out extensive measurement of natural ¹³C‐abundance (δ¹³C) in oil palm tissues, including fruits at different maturation stages. We find a ¹³C‐enrichment in heterotrophic organs compared to mature leaves, with roots being the most ¹³C‐enriched. The δ¹³C in fruits decreased during maturation, reflecting the accumulation in ¹³C‐depleted lipids. We further used observed δ¹³C values to compute plausible carbon fluxes using a steady‐state model of ¹³C‐distribution including metabolic isotope effects (¹²v/¹³v). The results suggest that fruits represent a major respiratory loss (≈39% of total tree respiration) and that sink organs such as fruits are fed by sucrose from leaves. That is, glucose appears to be a quantitatively important compound in palm tissues, but computations indicate that it is involved in dynamic starch metabolism rather that C‐exchange between organs.     

Development, characterisation, and across-taxa utility of oil palm (Elaeis guineensis Jacq.) microsatellite markers.

The results of the development of oil palm (Elaeis guineensis Jacq.) microsatellite markers are given step by step, from the screening of libraries enriched in (GA)n, (GT)n, and (CCG)n simple-sequence repeats (SSRs) to the final characterisation of 21 SSR loci. Also published are primer sequences, estimates of allele size range, and expected heterozygosity in E. guineensis and in the closely related species E. oleifera, in which an optimal utility of the SSR markers was observed. Multivariate data analyses showed the ability of SSR markers to efficiently reveal the genetic-diversity structure of the genus Elaeis in accordance with known geographical origins and with measured genetic relationships based on previous molecular studies. High levels of allelic variability indicated that E. guineensis SSRs will be a powerful tool for genetic studies of the genus Elaeis, including variety identification and intra- or inter-specific genetic mapping. PCR amplification tests on a subset of 16 other palm species and allele-sequence data showed that E. guineensis SSRs are putative transferable markers across palm taxa. In addition, phenetic information based on SSR flanking region sequences makes E. guineensis SSR markers a potentially useful molecular resource for any researcher studying the phylogeny of palm taxa.