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The CRISPR/Cas9 genome editing system has been in the spotlight compared to programmable nucleases such as ZFNs and TALENs due to its simplicity, versatility, and high efficiency. CRISPR/Cas9 has revolutionized plant genetic engineering and is broadly used to edit various plants' genomes, including those transformation-recalcitrant species such as oil palm. This review will comprehensively present the CRISPR-Cas9 system's brief history and underlying mechanisms. We then highlighted the establishment of the CRISPR/Cas9 system in plants with an emphasis on the strategies of highly efficient guide RNA design, the establishment of various CRISPR/Cas9 vector systems, approaches of multiplex editing, methods of transformation for stable and transient techniques, available methods for detecting and analyzing mutations, which have been applied and could be adopted for CRISPR/Cas9 genome editing in oil palm. In addition, we also provide insight into the strategy of DNA-free genome editing and its potential application in oil palm. [ABSTRACT FROM AUTHOR]

Background: Hemibiotrophic pathogen such as the fungal pathogen Ganoderma boninense that is destructive to oil palm, manipulates host defense mechanism by strategically switching from biotrophic to necrotrophic phase. Our previous study revealed two distinguishable expression profiles of oil palm genes that formed the basis in deducing biotrophic phase at early interaction which switched to necrotrophic phase at a later stage of infection. Results: The present report is a continuing study from our previous published transcriptomic profiling of oil palm seedlings against G. boninense. We focused on identifying differentially expressed genes (DEGs) encoding transcription factors (TFs) from the same RNA-seq data; resulting in 106 upregulated and 108 downregulated TFs being identified. The DEGs are involved in four established defense-related pathways responsible for cell wall modification, reactive oxygen species (ROS)-mediated signaling, programmed cell death (PCD) and plant innate immunity. We discovered upregulation of JUNGBRUNNEN 1 (EgJUB1) during the fungal biotrophic phase while Ethylene Responsive Factor 113 (EgERF113) demonstrated prominent upregulation when the palm switches to defense against necrotrophic phase. EgJUB1 was shown to have a binding activity to a 19 bp palindromic SNBE1 element, WNNYBTNNNNNNNAMGNHW found in the promoter region of co-expressing EgHSFC-2b. Further in silico analysis of promoter regions revealed co-expression of EgJUB1 with TFs containing SNBE1 element with single nucleotide change at either the 5th or 18th position. Meanwhile, EgERF113 binds to both GCC and DRE/CRT elements promoting plasticity in upregulating the downstream defense-related genes. Both TFs were proven to be nuclear-localized based on subcellular localization experiment using onion epidermal cells. Conclusion: Our findings demonstrated unprecedented transcriptional reprogramming of specific TFs potentially to enable regulation of a specific set of genes during different infection phases of this hemibiotrophic fungal pathogen. The results propose the intricacy of oil palm defense response in orchestrating EgJUB1 during biotrophic and EgERF113 during the subsequent transition to the necrotrophic phase. Binding of EgJUB1 to SNBE motif instead of NACBS while EgERF113 to GCC-box and DRE/CRT motifs is unconventional and not normally associated with pathogen infection. Identification of these phase-specific oil palm TFs is important in designing strategies to tackle or attenuate the progress of infection. [ABSTRACT FROM AUTHOR]

Key message: The oil palm EgPAL1 gene promoter and its regulatory region were functional as a promoter in the heterologous system of Arabidopsis according to the cis-acting elements present in that region. The promoter was developmentally regulated, vascular tissue specific and responsive to water stress agents. Abstract: Phenylalanine ammonia lyase (PAL, EC 4.3.1.24) is the key enzyme of the phenylpropanoid pathway which plays important roles in plant development and adaptation. To date, there is no report on the study of PAL from oil palm ( Elaeis guineensis), an economically important oil crop. In this study, the 5′ regulatory sequence of a highly divergent oil palm PAL gene ( EgPAL1) was isolated and fused with GUS in Arabidopsis to create two transgenic plants carrying the minimal promoter with (2302 bp) and without its regulatory elements (139 bp). The regulatory sequence contained cis-acting elements known to be important for plant development and stress response including the AC-II element for lignin biosynthesis and several stress responsive elements. The promoter and its regulatory region were fully functional in Arabidopsis. Its activities were characterised by two common fundamental features of PAL which are responsive to plant internal developmental programme and external factors. The promoter was developmentally regulated in certain organs; highly active in young organs but less active or inactive in mature organs. The presence of the AC elements and global activity of the EgPAL1 promoter in all organs resembled the property of lignin-related genes. The existence of the MBS element and enhancement of the promoter activity by PEG reflected the behaviour of drought-responsive genes. Our findings provide a platform for evaluating oil palm gene promoters in the heterologous system of Arabidopsis and give insights into the activities of EgPAL1 promoter in oil palm. [ABSTRACT FROM AUTHOR]

The protoplast system is robust and versatile for plant genetic engineering works. There are various ways to deliver nucleic acid materials into the protoplasts, but polyethylene glycol (PEG)-mediated method offers minimal damage compared to others. Therefore, an established PEG-mediated transformation protocol with optimised parameters is needed to improve the system's reproducibility and reliability. This study utilised oil palm mesophyll protoplasts as transformation cells and the red fluorescent protein (DsRED) as a visual reporter. Based on the transformation efficiency scored after 72 h, a method with the following parameters: 5 min incubation in 35% PEG, 10 min incubation with 50 µg DNA, and 90 s heat-shock treatment at 42 °C was found to be the best for oil palm protoplast PEG-mediated transfection. A significant improvement in transformation efficiency at about 56% was achieved using this method compared to the previous study (5%). This newly developed protocol was then used to evaluate the efficiency in terms of the relative strength of 7 promoters, including two plant constitutive promoters, three oil palm constitutive promoters, and two oil palm tissue-specific promoters in regulating the expression of the transgene in oil palm. Based on the results from three individual replicates, CaMV35S showed to be the most efficient amongst the tested promoters in driving the transgene expression in oil palm mesophyll protoplasts, followed by oil palm translationally control tumour protein (TCTP) and maize-ubiquitin (Ubi1) promoters. [Display omitted] • An efficient PEG-mediated transformation of oil palm mesophyll protoplasts. • High transformation efficiency compared to the previous protocol. • A reliable and reproducible oil palm protoplast-based transient gene expression assay. • The CaMV35S promoter was efficient for transgene expression in oil palm protoplasts. [ABSTRACT FROM AUTHOR]

Agricultural sorbents have received attention for their effectiveness in oil removal. In Malaysia, oil palm's empty fruit bunch (EFB) spikelets are an abundant agricultural waste that provides a non-toxic, renewable resource of cellulosic materials. In this study, the effectiveness of EFB spikelets to remove oil spills from seawater pollution in a filter system was investigated and the best optimisation approach for filtering conditions was determined. Experiments for oil spill clean-up were performed using a filter-based oil sorption system with a series of conditions such as temperature, time, packing density, and oil concentration to evaluate sorption capacity, oil and water absorbed efficiency. Fourier transform infrared spectroscopy (FTIR) was used to characterise the physicochemical properties of untreated and treated EFB fibres. Based on one-factor-at -a-time (OFAT) analysis conducted at 160 °C for 30 min on 0.1 g/cm3 of packing density containing 25% diesel, 8.667 mL of oil and 5 mL of water was absorbed. In response surface methodology (RSM), the three parameters of temperature, packing density and diesel concentration were observed as significant. From RSM fitting model analysis, the predicted value obtained for both oil and water absorbed were 8.805 and 5.213 mL, respectively. The experimental RSM values of 9 and 5 mL of oil and water absorbed were obtained. The result demonstrated the validity of the model as the experimental RSM values were close to the RSM model's prediction. As compared to OFAT, the RSM method is more efficient in oil removal. This research contributes to a better knowledge of the usage of a natural sorbent as a method of diesel pollution remediation. [ABSTRACT FROM AUTHOR]

Oil pollution such as diesel poses a significant threat to the environment. Due to this, there is increasing interest in using natural materials mainly from agricultural waste as organic oil spill sorbents. Oil palm's empty fruit bunch (EFB), a cost-effective material, non-toxic, renewable resource, and abundantly available in Malaysia, contains cellulosic materials that have been proven to show a good result in pollution treatment. This study evaluated the optimum screening part of EFB that efficiently absorbs oil and the physicochemical characterisation of untreated and treated EFB fibre using Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). The treatment conditions were optimised using one-factor-at-a-time (OFAT), which identified optimal treatment conditions of 170 °C, 20 min, 0.1 g/cm3, and 10% diesel, resulting in 23 mL of oil absorbed. The predicted model was highly significant in statistical Response Surface Methodology (RSM) and confirmed that all the parameters (temperature, time, packing density, and diesel concentration) significantly influenced the oil absorbed. The predicted values in RSM were 175 °C, 22.5 min, 0.095 g/cm3, and 10%, which resulted in 24 mL of oil absorbed. Using the experimental values generated by RSM, 175 °C, 22.5 min, 0.095 g/cm3, and 10%, the highest oil absorption achieved was 24.33 mL. This study provides further evidence, as the data suggested that RSM provided a better approach to obtain a high efficiency of oil absorbed. [ABSTRACT FROM AUTHOR]

• An efficient protocol for protoplasts isolation from in vitro leaf and mesocarp of oil palm has been established. • Enzymes composition effect on the yield and viability of protoplast are significant. • Scraping the waxy layer on oil palm leaf increased the protoplast isolation efficiency. • Vacuum treatment enhanced the penetration of enzymes into tissues. • 10% ficoll in 0.6 M mannitol improved the separation of protoplasts from impurities. The absence of a cell wall on the protoplast contributes to its versatility. Its flexibility for DNA manipulation and the possibility of rapid cell-based assay is desirable in the plant biotechnology field. This study was carried out to improve protoplast isolation from oil palm in vitro leaf and mesocarp tissues. The factors affecting protoplast isolation efficiency were optimized, including the protocols and enzyme composition involved, focusing on the oil palm in vitro leaf first. Incubation of oil palm leaf sample with an enzyme mixture of cellulase R-10, macerozyme R-10, driselase, and pectolyase Y-23, for 14 h has successfully produced up to 2.5 × 106 protoplasts g-1 fresh weight (FW)-1 with 95% viability. Incubation of oil palm mesocarp tissue with the optimized enzyme mixture for 2 h at static condition has also successfully produced 3.98 × 106 protoplasts g-1 FW-1 with 85% viability. Besides, it was found that increasing the sample's surface area in contact with enzyme solution by slicing the samples into narrow strips and thin layers has improved the penetration of enzymes into the tissues and enhanced the isolation efficiency. In addition, a plasmolysis step before enzymatic treatment has also improved the protoplast viability by minimizing the damage incurred during isolation. The successful isolation of protoplast from oil palm leaf and mesocarp has enabled the study of gene function and the characterization of endogenous tissue-specific promoter being carried out in vivo. [ABSTRACT FROM AUTHOR]

Basal stem rot (BSR) disease caused by pathogenic fungus Ganoderma boninense is a significant concern in the oil palm industry. G. boninense infection in oil palm induces defense-related genes. To understand oil palm defense mechanisms in response to fungal invasion, we analyzed differentially expressed genes (DEGs) derived from RNA-sequencing (RNA-seq) transcriptomic libraries of oil palm roots infected with G. boninense. A total of 126 DEGs were detected from the transcriptomic libraries of G. boninense-infected root tissues at different infection stages. Functional annotation via pathway enrichment analyses revealed that the DEGs were involved in the defense response against the pathogen. The expression of the selected DEGs was further confirmed using real-time quantitative PCR (qPCR) on independent oil palm seedlings and mature palm samples. Seven putative defense-related DEGs consistently showed upregulation in seedlings and mature plants during G. boninense infection. These seven genes might potentially be developed as biomarkers for the early detection of BSR in oil palm. [ABSTRACT FROM AUTHOR]

Inorganic phosphate (Pi) starvation is an important abiotic constraint that affects plant cellular homeostasis, especially in tropical regions with high acidic soil and less solubilizable Pi. In the current work, oil palm seedlings were hydroponically maintained under optimal Pi-supply and no Pi-supply conditions for 14 days, and metabolites were measured by gas chromatography–mass spectrometry (GC–MS), from leaves and roots, after seven and 14 days of treatment, to investigate biochemical pathways in relation to P-utilizing strategy. After seven days of limited Pi, plant leaves showed increased levels of most soluble sugars, and after 14 days, the sugars' level decrease, except for erythritol, mannose, fructose, and glucose, which showed the highest levels. Rather in root samples, there were different but overlapping alterations, mainly on sugars, amino acids, and organic acids. The leaf sample was shown to have the highest response of sugars with myo-inositol playing a vital role in the redistribution of sugars, while maltose levels increased, indicating active degradation of starch in the root. High levels of glycerol and stearate in both roots and leaves suggest the metabolism of storage lipids for cellular energy during Pi-deficient conditions. [ABSTRACT FROM AUTHOR]

• First report on the use of Discosoma red fluorescent protein (DsRED) - as visual reporter marker in oil palm cells. • Seven DsRED gene vectors driven by a different promoter were constructed and available for the scientific community. • The expression of DsRED in oil palm cells exhibited better photostability and brightness compared to GFP. Genetic modification of oil palm is being carried out to explore new avenues for improving and expanding the potential of oil palm. Multiple bottlenecks in delivering the DNA into oil palm cells and generating the transgenic oil palm required us to look into an alternative marker which could help us assess the efficiency of the transformation system, moreover at the early stage considering that oil palm is a slow growing plant. In this study, we have constructed seven transformation vectors carrying a gene encoding DsRED, each driven by a different promoter, i.e. plant constitutive promoter, oil palm constitutive promoter or oil palm tissue-specific promoter. The vectors were transformed into oil palm embryogenic calli via microprojectile bombardment and the red fluorescence signals were observed after 48 h. Based on the observation, the construct driven by the CaMV35S promoter produced the highest number of red fluorescent spots. The oil palm calli bombarded with pUbi-DsRED plasmid suffered the most number of red fluorescent spots losses from day three until day 42, as compared to other constructs. The red fluorescent signals of DsRED produced across all transformed samples appeared to be bright and very distinctive as compared to the background. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 is a powerful genome editing tool applicable to diverse plant species. However, the application of CRISPR/Cas9-ribonucleoproteins (RNPs) in oil palm (Elaeis guineensis) has not yet been reported. In this paper, the specificity, efficiency, and sensitivity of CRISPR/Cas9-RNPs in oil palm were evaluated by assessing the effect of different amounts of Cas9 and heat stress using E. guineensis phytoene desaturase (EgPDS) as the target gene. Seven gRNAs were designed and combined with Cas9 protein to form three combinations of RNPs complexes; RNP1/RNP2, RNP3/RNP4, and RNP5/RNP6/RNP7. Assessment of optimal amount of Cas9 and heat stress using RNP5/RNP6/RNP7 demonstrated that 40 µg of Cas9 and heat treatment at 39ºC increased the genome editing efficiency up to 95% in oil palm protoplasts. The use of both optimal conditions produced targeted mutations at frequencies of up to 63.6% in protoplasts transformed with RNP1/RNP2 and RNP3/RNP4 and up to 100% with RNP5/RNP6/RNP7 combinations. Multiple site mutagenesis at two to three target sites with fragment removal up to 179 bp, 69 bp, and 816 bp occurred at high frequencies producing editing efficiency and a Knockout Score (KO) of 100%. Altogether, this study demonstrates a promising, highly efficient, and precise transgene-free CRISPR/Cas9-RNPs genome editing platform in oil palm that also holds enormous potential for application in other transformation-recalcitrant species. [Display omitted] • Successful delivery of multiple ribonucleoproteins into oil palm protoplasts. • Optimum amount of Cas9 increases the CRISPR/Cas9-RNPs gene editing efficiency. • Heat stress produces a high CRISPR/Cas9-RNPs mutation rate in oil palm protoplasts. • CRISPR/Cas9-RNPs system produces 100% mutation frequency in oil palm protoplasts. [ABSTRACT FROM AUTHOR]

Basal stem rot (BSR) is a detrimental disease in oil palm (Elaeis guineensis) due to the host's lack of symptoms during the early phase of infection. Early detection of BSR at non-symptomatic phase is feasible via molecular-based approaches and has become the major interest among researchers. Our previous study had identified a differentially expressed gene encoding auxin efflux carrier component 5-like (EgPIN5) at 11 day-post-inculation of Ganoderma boninense (symptomless early stage of infection) on oil palm seedlings. We further evaluated the gene expression on samples collected from an oil palm plantation at different BSR stages (symptomless, mild and severe). Our findings revealed that EgPIN5 was expressed in symptomless group but was not expressed in either mild or severe BSR groups. Next, we developed a sandwich-type aptamer-based lateral flow assay (LFA) against EgPIN5 RNA by adapting the versatile DNA-functionalized gold nanoparticles for colorimetric detection. The LFA strip demonstrated visible colorimetric signal of EgPIN5 RNA at 12.5 ng/μL with limit of detection at 2.45 ng/μL using a scanning device. Based on our consistent results, we presented EgPIN5 as a diagnostic biomarker for symptomless BSR infection in oil palm. Utilization of LFA assay strip for practical EgPIN5 RNA detection is hoped to encourage establishment of a fast, cheap, and sensitive point-of-care detection system in plant biology. • Induced expression of EgPIN5 was observed at early stage of Ganoderma boninense infection of oil palm. • Aptamer-based biosensor was developed with high specificity in detecting EgPIN5 RNA. • Sensitivity of detection on a lateral flow strip was as low as 2.45 ng/uL. [ABSTRACT FROM AUTHOR]

Modification of lipid composition in the mesocarp tissue of oil palm involves genetic manipulation of multiple genes. More than one mesocarp-preferential promoter is necessary for the expression of individual transgenes in the same plant to obviate transcriptional gene silencing. This study aimed to identify genes that are preferentially expressed in the mesocarp tissue and characterize selected candidate mesocarp-preferential promoters. Ten transcripts that were preferentially expressed in the mesocarp tissue were identified from the analysis of 82 transcriptome datasets of 12 different oil palm tissues. The expression of two candidate genes, MSP-C1 and MSP-C6 , was verified to be preferentially expressed in the mesocarp tissues and shown to have a low expression level in non-mesocarp tissues by reverse transcription quantitative real-time PCR (RT-qPCR). MSP-C6 promoter fragments of different lengths were transformed into tomato plants for further characterization. Both unripe and ripe fruits of transgenic tomato plants transformed with a construct harboring the MSP-C6-F1 (2014 bp) promoter were shown to have high beta-glucuronidase (GUS) activities. The findings of this study suggest the potential applications of the MSP-C6 promoter as a molecular tool for genetic engineering of novel traits in fruit crops. • Ten mesocarp-preferential transcripts were identified from the analysis of 82 oil palm transcriptome datasets. • Two candidate genes were verified to be preferentially expressed in the oil palm mesocarp tissues. • The MSP-C6-F1 (2014 bp) promoter fragment was shown to be able to drive preferential gene expression in tomato fruits. • Characterization of mesocarp-specific promoters contribute to future genetic manipulation of oil synthesis in oil palm. [ABSTRACT FROM AUTHOR]

Oil palm is subjected to various diseases including Basal Stem Rot (BSR) which is mainly caused by the basidiomycetes fungi, Ganoderma. However, studies on the fungal infection mechanism especially at molecular level and the biological processes involved are still very limited. Fungal cyclophilin (CYP) has been reported to be involved in fungal pathogenicity but involvement of CYP in the pathogenicity of Ganoderma boninense has not been revealed yet. The main objective of this study is to isolate cDNAs encoding CYP and to profile the expression level of these transcripts during infection stages of G. boninense. In this study, five full-length cDNAs encoding CYP were successfully isolated from G. boninense . They were classified as different family members of CYP because significant differences could be observed on their coding, 5′ or 3′ un-translated regions (UTRs). The expression of these five CYP transcripts in different type of G. boninense tissues and the infecting mycelia tissues was studied using real time quantitative PCR (qPCR). Based on the expression results and analysis, the potential functions of these CYP transcripts were predicted to be involved in the development of fruiting body (GbCYP201) and fungal stress response or pathogenicity (GbCYP203 and GbCYP205). [ABSTRACT FROM AUTHOR]