Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.
Enroute to mapping QTLs for yield components in oil palm, we constructed the linkage map of a FELDA high yielding oil palm (Elaeis guineensis), hybrid cross. The parents of the mapping population are a Deli dura and a pisifera of Yangambi origin. The cross out-yielded the average by 8-21% in four trials all of which yielded comparably to the best current commercial planting materials. The higher yield derived from a higher fruit oil content. SSR markers in the public domain - from CIRAD and MPOB, as well as some developed in FELDA - were used for the mapping, augmented by locally-designed AFLP markers. The female parent linkage map comprised 317 marker loci and the male parent map 331 loci, both in 16 linkage groups each. The number of markers per group ranged from 8-47 in the former and 12-40 in the latter. The integrated map was 2,247.5 cM long and included 479 markers and 168 anchor points. The number of markers per linkage group was 15-57, the average being 29, and the average map density 4.7 cM. The linkage groups ranged in length from 77.5 cM to 223.7 cM, with an average of 137 cM. The map is currently being validated against a closely related population and also being expanded to include yield related QTLs.
Abscisic acid (ABA) is an important phytohormone involved in the abiotic stress resistance in plants. The ABA-responsive element (ABRE) binding factors play significant roles in the plant development and response to abiotic stresses, but none so far have been isolated and characterized from the oil palm. Two ABA-responsive cDNA clones, named EABF and EABF1, were isolated from the oil palm fruits using yeast one-hybrid system. The EABF had a conserved AP2/EREBP DNA-binding domain (DNA-BD) and a potential nuclear localization sequence (NLS). No previously known DNA-BD was identified from the EABF1 sequence. The EABF and EABF1 proteins were classified as DREB/CBF and bZIP family members based on the multiple sequence alignment and phylogenetic analysis. Both proteins showed ABRE-binding and transcriptional activation properties in yeast. Furthermore, both proteins were able to trans-activate the down-stream expression of the LacZ reporter gene in yeast. An electrophoretic mobility shift assay revealed that in addition to the ABRE sequence, both proteins could bind to the DRE sequence as well. Transcriptional analysis revealed that the expression of EABF was induced in response to the ABA in the oil palm fruits and leaves, but not in roots, while the EABF1 was constitutively induced in all tissues. The expressions of both genes were strongly induced in fruits in response to the ABA, ethylene, methyl jasmonate, drought, cold and high-salinity treatments, indicating that the EABF and EABF1 might act as connectors among different stress signal transduction pathways. Our results indicate that the EABF and EABF1 are novel stress-responsive transcription factors, which are involved in the abiotic stress response and ABA signaling in the oil palm and could be used for production of stress-tolerant transgenic crops.
RNA-binding proteins (RBPs) have been implicated as regulatory proteins involved in the post-transcriptional processes of gene expression in plants under various stress conditions. In this study, we report the cloning and characterization of a gene, designated as EgRBP42, encoding a member of the plant heterogeneous nuclear ribonucleoprotein (hnRNP)-like RBP family from oil palm (Elaeis guineensis Jacq.). EgRBP42 consists of two N-terminal RNA recognition motifs and a glycine-rich domain at the C-terminus. The upstream region of EgRBP42 has multiple light-responsive, stress-responsive regulatory elements and regulatory elements associated with flower development. Real-time RT-PCR analysis of EgRBP42 showed that EgRBP42 was expressed in oil palm tissues tested, including leaf, shoot apical meristem, root, female inflorescence, male inflorescence and mesocarp with the lowest transcript level in the roots. EgRBP42 protein interacted with transcripts associated with transcription, translation and stress responses using pull-down assay and electrophoretic mobility shift assay. The accumulation of EgRBP42 and its interacting transcripts were induced by abiotic stresses, including salinity, drought, submergence, cold and heat stresses in leaf discs. Collectively, the data suggested that EgRBP42 is a RBP, which responds to various abiotic stresses and could be advantageous for oil palm under stress conditions. Key message EgRBP42 may be involved in the post-transcriptional regulation of stress-related genes important for plant stress response and adaptation.
Glucanases are enzymes that hydrolyze a variety β-d-glucosidic linkages. Plant β-1,3-glucanases are able to degrade fungal cell walls; and promote the release of cell-wall derived fungal elicitors. In this study, three full-length cDNA sequences encoding oil palm (Elaeis guineensis) glucanases were analyzed. Sequence analyses of the cDNA sequences suggested that EgGlc1-1 is a putative β-d-glucan exohydolase belonging to glycosyl hydrolase (GH) family 3 while EgGlc5-1 and EgGlc5-2 are putative glucan endo-1,3-β-glucosidases belonging to GH family 17. The transcript abundance of these genes in the roots and leaves of oil palm seedlings treated with Ganoderma boninense and Trichoderma harzianum was profiled to investigate the involvement of these glucanases in oil palm during fungal infection. The gene expression of EgGlc1-1 in the root of oil palm seedlings was increased by T. harzianum but suppressed by G. boninense; while the gene expression of both EgGlc5-1 and EgGlc5-2 in the roots of oil palm seedlings was suppressed by G. boninense or/and T. harzianum.
We evaluated 38 dura x pisifera (DP) oil palm progenies in four locations in Malaysia for genotype by environment interaction and genotypic stability studies. The DP progenies derived from crosses between pisifera palms of AVROS, Serdang S27B, Serdang 29/36, and Lever Cameroon were chosen to be the males' parent and Deli dura palms designated as females' parent. All the locations differed in terms of soil physical and chemical properties, and the soil types ranged from coastal clay to inland soils. The genotype by environment interaction and stability of the individual genotypes were analyzed for oil yield trait using several stability techniques. A genotype by environment interaction was detected for oil yield and it had a larger variance component than genotypic variance (σ(2)(gl)/σ(2)(g) = 139.7%). Genotype by environment interaction of oil yield was largely explained by a non-linear relationship between genotypic and environmental values. Overall assessment of individual genotypic stability showed that seven genotypes were highly stable and had consistent performance over the environments for the oil yield trait [total individual genotype stability scored more than 10 and mean oil yielded above the average of the environment (genotype means are more than 34.37 kg·palm(-1)·year(-1))]. These genotypes will be useful for oil palm breeding and tissue culture programs for developing high oil yielding planting materials with stable performance.
The performance of 11 oil palm AVROS (Algemene Vereniging van Rubberplanters ter Oostkust van Sumatra) pisiferas was evaluated based on their 40 dura x pisifera (DxP) progenies tested on inland soils, predominantly of Serdang Series. Fresh fruit bunch (FFB) yield of each pisiferas ranged from 121.93 to 143.9 kg palm⁻¹ yr⁻¹ with trial mean of 131.62 kg palm⁻¹ yr⁻¹. Analysis of variance (ANOVA) showed low genetic variability among pisifera parents for most of the characters indicating uniformity of the pisifera population. This was anticipated as the AVROS pisiferas were derived from small population and were inbred materials. However, some of the pisiferas have shown good general combining ability (GCA) for certain important economic traits. Three pisiferas (P1 (0.174/247), P3 (0.174/498), P11 (0.182/308)) were identified of having good GCA for FFB yield while pisiferas P1 (0.174/247), P10 (0.182/348), and P11 (0.182/308) were good combiners for oil-to-bunch ratio (O/B). The narrow genetic base of these materials was the main obstacle in breeding and population improvement. However, efforts have been made to introgress this material with the vast oil palm germplasm collections of MPOB for rectifying the problem.
Basal stem rot (BSR) disease caused by the fungus Ganoderma boninense is the most serious disease affecting the oil palm; this is because the disease escapes the early disease detection. The biocontrol agent Trichoderma harzianum can protect the disease only at the early stage of the disease. In the present study, the expression levels of three oil palm (Elaeis guineensis Jacq.) chitinases encoding EgCHI1, EgCHI2, and EgCHI3 at 2, 5, and 8 weeks inoculation were measured in oil palm leaves from plants treated with G. boninense or T. harzianum alone or both.
Species-specific simple sequence repeat (SSR) markers are favored for genetic studies and marker-assisted selection (MAS) breeding for oil palm genetic improvement. This report characterizes 20 SSR markers from an Elaeis oleifera genomic library (gSSR). Characterization of the repeat type in 2000 sequences revealed a high percentage of di-nucleotides (63.6%), followed by tri-nucleotides (24.2%). Primer pairs were successfully designed for 394 of the E. oleifera gSSRs. Subsequent analysis showed the ability of the 20 selected E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The average Polymorphism Information Content (PIC) value for the SSRs was 0.402, with the tri-repeats showing the highest average PIC (0.626). Low values of observed heterozygosity (H(o)) (0.164) and highly positive fixation indices (F(is)) in the E. oleifera germplasm collection, compared to the E. guineensis, indicated an excess of homozygosity in E. oleifera. The transferability of the markers to closely related palms, Elaeis guineensis, Cocos nucifera and ornamental palms is also reported. Sequencing the amplicons of three selected E. oleifera gSSRs across both species and palm taxa revealed variations in the repeat-units. The study showed the potential of E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The markers are also a valuable genetic resource for studying E. oleifera and other genus in the Arecaceae family.
Chitinases are glycosyl hydrolases that cleave the β-1,4-glycosidic linkages between N-acetylglucosamine residues in chitin which is a major component of fungal cell wall. Plant chitinases hydrolyze fungal chitin to chitin oligosaccharides that serve as elicitors of plant defense system against fungal pathogens. However, plants synthesize many chitinase isozymes and some of them are not pathogenesis-related. In this study, three full-length cDNA sequences encoding a putative chitinase (EgChit3-1) and two chitinase-like proteins (EgChit1-1 and EgChit5-1) have been cloned from oil palm (Elaeis guineensis) by polymerase chain reaction (PCR). The abundance of these transcripts in the roots and leaves of oil palm seedlings treated with Ganoderma boninense (a fungal pathogen) or Trichoderma harzianum (an avirulent symbiont), and a combination of both fungi at 3, 6 and 12 weeks post infection were profiled by real time quantitative reverse-transcription (qRT)-PCR. Our findings showed that the gene expression of EgChit3-1 increased significantly in the roots of oil palm seedlings treated with either G. boninense or T. harzianum and a combination of both; whereas the gene expression of EgChit1-1 in the treated roots of oil palm seedlings was not significantly higher compared to those of the untreated oil palm roots. The gene expression of EgChit5-1 was only higher in the roots of oil palm seedlings treated with T. harzianum compared to those of the untreated oil palm roots. In addition, the gene expression of EgChit1-1 and EgChit3-1 showed a significantly higher gene expression in the leaf samples of oil palm seedlings treated with either G. boninense or T. harzianum.
Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are involved in cell growth regulation, transportation of metal ions and detoxification of heavy metals. A mesocarp-specific metallothionein-like gene (MT3-A) promoter was isolated from the oil palm (Elaeis guineensis Jacq). A vector construct containing the MT3-A promoter fused to the β-glucuronidase (GUS) gene in the pCAMBIA 1304 vector was produced and used in Agrobacterium-mediated transformation of tomato. Histochemical GUS assay of different tissues of transgenic tomato showed that the MT3-A promoter only drove GUS expression in the reproductive tissues and organs, including the anther, fruit and seed coat. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS mRNA and enzyme activity in the transgenic tomato (T(0)). No GUS mRNA was found in roots and leaves of transgenic tomato. In contrast, the leaves of transgenic tomato seedlings (T(1)) produced the highest GUS activity when treated with 150 μM Cu(2+) compared to the control (without Cu(2+)). However, Zn(2+) and Fe(2+) treatments did not show GUS expression in the leaves of the transgenic tomato seedlings. Interestingly, the results showed a breaking-off tissue-specific activity of the oil palm MT3-A promoter in T(1) seedlings of tomato when subjected to Cu(2+) ions.
Oil palm is the most productive oil-bearing crop. Although it is planted on only 5% of the total world vegetable oil acreage, palm oil accounts for 33% of vegetable oil and 45% of edible oil worldwide, but increased cultivation competes with dwindling rainforest reserves. We report the 1.8-gigabase (Gb) genome sequence of the African oil palm Elaeis guineensis, the predominant source of worldwide oil production. A total of 1.535 Gb of assembled sequence and transcriptome data from 30 tissue types were used to predict at least 34,802 genes, including oil biosynthesis genes and homologues of WRINKLED1 (WRI1), and other transcriptional regulators, which are highly expressed in the kernel. We also report the draft sequence of the South American oil palm Elaeis oleifera, which has the same number of chromosomes (2n = 32) and produces fertile interspecific hybrids with E. guineensis but seems to have diverged in the New World. Segmental duplications of chromosome arms define the palaeotetraploid origin of palm trees. The oil palm sequence enables the discovery of genes for important traits as well as somaclonal epigenetic alterations that restrict the use of clones in commercial plantings, and should therefore help to achieve sustainability for biofuels and edible oils, reducing the rainforest footprint of this tropical plantation crop.
A key event in the domestication and breeding of the oil palm Elaeis guineensis was loss of the thick coconut-like shell surrounding the kernel. Modern E. guineensis has three fruit forms, dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), a hybrid between dura and pisifera. The pisifera palm is usually female-sterile. The tenera palm yields far more oil than dura, and is the basis for commercial palm oil production in all of southeast Asia. Here we describe the mapping and identification of the SHELL gene responsible for the different fruit forms. Using homozygosity mapping by sequencing, we found two independent mutations in the DNA-binding domain of a homologue of the MADS-box gene SEEDSTICK (STK, also known as AGAMOUS-LIKE 11), which controls ovule identity and seed development in Arabidopsis. The SHELL gene is responsible for the tenera phenotype in both cultivated and wild palms from sub-Saharan Africa, and our findings provide a genetic explanation for the single gene hybrid vigour (or heterosis) attributed to SHELL, via heterodimerization. This gene mutation explains the single most important economic trait in oil palm, and has implications for the competing interests of global edible oil production, biofuels and rainforest conservation.
Basal stem rot (BSR) is a major disease of oil palm caused by a pathogenic fungus, Ganoderma boninense. However, the interaction between the host plant and its pathogen is not well characterized. To better understand the response of oil palm to G. boninense, transcript profiles of eleven putative defence-related genes from oil palm were measured by quantitative reverse-transcription (qRT)-PCR in the roots of oil palms treated with G. boninense from 3 to 12 weeks post infection (wpi). These transcripts encode putative Bowman-Birk serine protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early methionine-labeled polypeptides (EgEMLP1 and 2), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein (EgMT), pathogenesis-related-1 protein (EgPRP), and type 2 ribosome-inactivating protein (EgT2RIP). The transcript abundance of EgBBI2 increased in G. boninense-treated roots at 3 and 6wpi compared to those of controls; while the transcript abundance of EgBBI1, EgDFS, EgEMLP1, EgMT, and EgT2RIP increased in G. boninense-treated roots at 6 or 12wpi. Meanwhile, the gene expression of EgDHN was up-regulated at all three time points in G. boninense-treated roots. The expression profiles of the eleven transcripts were also studied in leaf samples upon inoculation of G. boninense and Trichoderma harzianum to identify potential biomarkers for early detection of BSR. Two candidate genes (EgEMLP1 and EgMT) that have different profiles in G. boninense-treated leaves compared to those infected by T. harzianum may have the potential to be developed as biomarkers for early detection of G. boninense infection.
Clonal reproduction of oil palm by means of tissue culture is a very inefficient process. Tissue culturability is known to be genotype dependent with some genotypes being more amenable to tissue culture than others. In this study, genetic linkage maps enriched with simple sequence repeat (SSR) markers were developed for dura (ENL48) and pisifera (ML161), the two fruit forms of oil palm, Elaeis guineensis. The SSR markers were mapped onto earlier reported parental maps based on amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers. The new linkage map of ENL48 contains 148 markers (33 AFLPs, 38 RFLPs and 77 SSRs) in 23 linkage groups (LGs), covering a total map length of 798.0 cM. The ML161 map contains 240 markers (50 AFLPs, 71 RFLPs and 119 SSRs) in 24 LGs covering a total of 1,328.1 cM. Using the improved maps, two quantitative trait loci (QTLs) associated with tissue culturability were identified each for callusing rate and embryogenesis rate. A QTL for callogenesis was identified in LGD4b of ENL48 and explained 17.5% of the phenotypic variation. For embryogenesis rate, a QTL was detected on LGP16b in ML161 and explained 20.1% of the variation. This study is the first attempt to identify QTL associated with tissue culture amenity in oil palm which is an important step towards understanding the molecular processes underlying clonal regeneration of oil palm.
To better understand lipid biosynthesis in oil palm mesocarp, in particular the differences in gene regulation leading to and including de novo fatty acid biosynthesis, a multi-platform metabolomics technology was used to profile mesocarp metabolites during six critical stages of fruit development in comparatively high- and low-yielding oil palm populations. Significantly higher amino acid levels preceding lipid biosynthesis and nucleosides during lipid biosynthesis were observed in a higher yielding commercial palm population. Levels of metabolites involved in glycolysis revealed interesting divergence of flux towards glycerol-3-phosphate, while carbon utilization differences in the TCA cycle were proven by an increase in malic acid/citric acid ratio. Apart from insights into the regulation of enhanced lipid production in oil palm, these results provide potentially useful metabolite yield markers and genes of interest for use in breeding programmes.
Mantled fruits as a result of somaclonal variation are often observed from the oil palm plantlets regenerated via tissue culture. The mantling of fruits with finger-like and thick outer coating phenotypes significantly reduces the seed size and oil content, posing a threat to oil palm planters, and may jeopardize the economic growth of countries that depend particularly on oil palm plantation. The molecular aspects of the occurrence of somaclonal variations are yet to be known, possibly due to gene repression such as DNA methylation, histone methylation and histone deacetylation. Histone deacetylases (HDACs), involved in eukaryotic gene regulation by catalyzing the acetyl groups are removal from lysine residues on histone, hence transcriptionally repress gene expression. This paper described the total protein polymorphism profiles of somaclonal variants of oil palm and the effects of histone deacetylation on this phenomenon. Parallel to the different phenotypes, the protein polymorphism profiles of the mantled samples (leaves, fruits, and florets) and the phenotypically normal samples were proven to be different. Higher HDAC activity was found in mantled leaf samples than in the phenotypically normal leaf samples, leading to a preliminary conclusion that histone deacetylation suppressed gene expression and contributed to the development of somaclonal variants.
Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance.
The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels.
Twelve introgressed oil palm (Elaeis guineensis) progenies of Nigerian dura x Deli dura were evaluated for bunch yield, yield attributes, bunch quality components and vegetative characters at the Malaysian Palm Oil Board Research Station, in Keratong, Pahang, Malaysia. Analysis of variance revealed significant to highly significant genotypic differences, indicating sufficient genetic variability among the progenies for bunch yield and its attributes, vegetative characters and bunch quality components, except fruit to bunch ratio. Fresh fruit bunch yield ranged from 167 kg·palm(-1)·year(-1) in PK1330 to 212 kg·palm(-1)·year(-1) in PK1351, with a mean yield of 192 kg·palm(-1)·year(-1). Among the progeny, PK1313 had the highest oil to bunch ratio (19.36%), due to its high mesocarp to fruit ratio, fruit to bunch ratio and low shell to fruit ratio. Among the progenies, PK1313 produced the highest oil yield of 31.4 kg·palm(-1)·year(-1), due to a high mesocarp to fruit ratio (61.2%) and a low shell to fruit ratio (30.7%), coupled with high fruit to bunch ratio (65.6%). PK1330 was found promising for selection, as it had desirable vegetative characters, including smaller petiole cross section (27.15 cm2), short rachis length (4.83 m), short palm height (1.85 m), and the lowest leaf number (164.6), as these vegetative characters are prerequisites for selecting palms for high density planting and high yield per hectare. The genetic variability among the progenies was found to be high, indicating ample scope for further breeding, followed by selection.