Category Archives: 2019 Vol 31 June

ISOLATION OF HIGH QUALITY TOTAL RNA FROM VARIOUS TISSUES OF OIL PALM (Elaeis guineensis) FOR REVERSE TRANSCRIPTION QUANTITATIVE REAL-TIME PCR (RT-qPCR)

The extraction of high quality total ribonucleic acid (RNA) has become an essential step for downstream plant molecular biology research. In oil palm, several total RNA extraction protocols have been reported across specific tissues using either conventional method or commercial kits. To our knowledge, there is no specific total RNA extraction method described for oil palm whole fruit which contains lipids, polysaccharides, protein, polyphenols and other secondary metabolites. Here, a modified cetyltrimethylammonium bromidelithium chloride (CTAB-LiCl) method with addition of phenol was established to extract total RNA across various oil palm tissues, especially for whole fruit at eight weeks after anthesis (WAA) and 15 WAA. To assess the extracted total RNA, quality and quantity as well as integrity were evaluated using spectrophotometer and bioanalyser, respectively. It was found that modified CTAB-LiCl method with addition of phenol was able to produce good quality (both A260/A280 and A260/A230 ratios > 1.8) and intact (RIN > 7.4) total RNA. Furthermore, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was successfully performed across all total RNA extracted using two reference genes (PD00569 and pOPEA01332) and one gene of interest, ethylene-responsive transcription factor-3 like, designated as PD00088. The results indicated that the method is suitable for extraction of total RNA from whole fruit as well as other oil palm tissues and is amenable to RT-qPCR.

IDENTIFICATION OF RELIABLE REFERENCE GENES FOR GENE EXPRESSION STUDIES OF OIL PALM PLANTLETS USING NORMFINDER AND BESTKEEPER ALGORITHMS

Gene expression profiling analysis using real-time quantitative polymerase chain reaction (qRT-PCR) is commonly used due to its reliability and accuracy. Gene expression profiles of particular genes in different tissues and developmental stages aid in the understanding of their biological functions. However, the accuracy and reliability of qRT-PCR to study gene expression demand the use of species-specific reference genes for normalisation. Therefore, appropriate reference genes for qRT-PCR need to be determined for oil palm ( Elaeis guineensis) tissues. In this study, six candidate reference genes namely actin, Cyp2, GRAS, EgEfa1, F-box, and GLU were examined in leaf, node, internode, root, cabbage, shoot primordial and basal stem tissues of two-year old clonal oil palms. Two statistical algorithms, NormFinder and BestKeeper verified the stability of GRAS and Cyp2 reference genes in the oil palm tissues tested. The combination of Cyp2 and GRAS reference genes are recommended for gene expression normalisation profiles in nurserystage oil palm plantlets.

MUTAGENIC EFFECTS OF GAMMA IRRADIATION ON OIL PALM (Elaeis guineensis Jacq.) SEEDLING GERMINATION AND GROWTH

Mutation induction has been used to generate genetic variability in most crop plants. This research was conducted to assess genetic variation induced by gamma radiation on the various treatment designated as M1, M2, M2M1 at 10 Gy. A randomised complete block design with four replicates was used for the experiment. Parameters such as percentage germination, root length, leaf area, plant height and stem/trunk circumference were taken from 4- to 12-month seedlings after planting. Data was analysed using GenStat (12th edition), which revealed significant differences in germination and growth of these oil palm seedlings. The study shows that percentage germination as well as growth parameters were stimulated in the M1 seedlings but inhibited in the M2 and M2M1 populations.

cDNA-SSR MARKERS FOR MOLECULAR EPIDEMIOLOGY OF Ganoderma boninense

Basal stem rot (BSR) caused by Ganoderma boninense is the most devastating and yield-limiting disease for the oil palm industry in South-east Asia. Information on the population biology of G. boninense for testing important hypotheses concerning BSR infection is lacking. This article describes the development of the most discerning molecular marker to date, i.e. cDNA-SSR, in an attempt to evaluate the genetic diversity and epidemiology of G. boninense. The Belitung Island in Indonesia was chosen for this study because it has BSR disease spread in first generation plantings of oil palm, and geographically not connected to the Sumatra main island. There are considerable field experiences alongside the indepth genetic analyses presented here. The basidiocarps of Ganoderma collected from BSR and upper stem rot (USR) infected oil palm were confirmed as G. boninense by phylogenetic analysis. The marker data inferred that the G. boninense isolates were very diverse, with heterozygosity of 0.777, reflecting random mating and the outcrossing nature of this fungus. Clonal spread was not found in the present study and isolates were genetically different, from palm to palm. The hierarchical cluster analysis and distance-standardised principal coordinates analysis suggested G. boninense spread from the coastal region (north) to inland areas (south) on Belitung Island. Despite the very small sample size, the thorough analyses revealed major shortcomings and difficuties in developing a set of polymorphic cDNA-SSR markers for epidemiology of G. boninense.

PHYTOL-CONTAINING SEAWEED EXTRACTS AS CONTROL FOR Ganoderma boninense

Basal stem rot (BSR) is a disease in oil palm caused by a fungal pathogen, Ganoderma boninense. Utilisation of seaweeds as a control agent has not been explored. This study investigated the anti-fungal potential of Malaysian seaweed extracts against G. boninense and identification of the compounds. Seaweeds cf Sargassum oligocystum, Caulerpa racemosa, Caulerpa racemosa var. lamouroxii and cfHalimeda macrophysa were collected and subjected to crude extraction with various solvents. Methanolic extracts of all species displayed the highest yield with an average of 15.25% compared to dry weight. Anti-fungal assays were carried out against G. boninense using the poisoned food technique and three highest inhibitions were exhibited by C. racemosa var. lamouroxii dichloromethane extract (46.82%), cf H. macrophysa dichloromethane extract (33.49%) and C. racemosa methanol extract (28.06%). Dominant compounds detected via gas chromatography-mass chromatography (GC-MS) in extracts with anti fungal potential includes phytol. Anti-fungal assay using standard phytol showed growth inhibition of G. boninense of up to 21% inhibition. Caulerpa racemosa var. lamouroxii, cf H. macrophysa, C. racemosa and cf S. oligocystum dichloromethane extracts contain 474 117, 106 and 19 mg litre-1 of phytol respectively. These findings suggested that Malaysian seaweeds are a good source of anti-fungal compounds for utilisation in controlling the BSR disease of oil palm in Malaysia.

PYRACLOSTROBIN SUPPRESSED Ganoderma BASAL STEM ROT (BSR), PROMOTED PLANT GROWTH AND INDUCED EARLY EXPRESSION OF β-1,3-GLUCANASE IN OIL PALM (Elaeis guineensis)

Pyraclostrobin, a new group of fungicide, was evaluated on its efficacy to suppress G. boninense as well as its effect on oil palm physiology, growth promotion, and induction of defense-related gene, β-1,3-glucanase. Hexaconazole was also used as a positive control. In vitro, the best inhibitory result was achieved using pyraclostrobin at 0.75 μg a.i. ml-1 with percentage inhibition of radial growth (PIRG) of 80%, followed by pyraclostrobin at 0.50 μg a.i. ml-1 (PIRG 73%) and hexaconazole at 0.15 μg a.i. ml-1 (PIRG 73%). Probit analysis also indicated that emulsifiable concentrate (EC) values of pyraclostrobin towards G. boninense mycelial growth were 0.25 μg a.i. ml-1 for EC50 and 0.58 μg a.i. ml-1 for EC75, which were further tested in vivo. In general, in vivo results indicated that the oil palm seedlings treated with pyraclostrobin at EC75 had significantly lower basal stem rot (BSR) infection, higher plant growth, and positive effects on plant physiology compared to pyraclostrobin at EC50 and were comparable to hexaconazole. Moreover, gene expression of β-1,3-glucanase in the seedlings treated with pyraclostrobin at EC75 indicated the highest level at the early stage. These results suggest that pyraclostrobin at EC75 was able to suppress BSR with high efficacy and significantly improved plant growth.

INTEGRATION OF LATEX TIMBER CLONE RUBBER AFFECTS THE OIL PALM YIELD

The dual purpose of latex timber clone (LTC) rubber; i.e. to produce good quality timber as well as latex yield, had inspired the Malaysian Palm Oil Board (MPOB) to carry out a study on integration of LTC with oil palm. Experiments were conducted to determine the suitability of integrating LTC (RRIM 2024 and RRIM 2025) with oil palm with respect to the effects on the yield of oil palm. The LTC were planted in the double avenue oil palm planting system at two planting densities of 136 palms ha-1 and 120 palms ha-1. In each oil palm planting density, LTC was planted at two different densities, 372 trees ha-1 and 422 trees ha-1. The advantage of double avenue oil palm planting systems is that the avenues between the palm rows are wider to allow for greater light penetration and better environment for growth of LTC. The study showed that both types of LTC, the RRIM 2024 and RRIM 2025 were growing well under the double avenue oil palm planting system. The rubber trees produced cup lumps and rubber wood log with no difference in the two oil palm planting densities. More importantly, this study found that the integration of LTC has affected the yield of oil palm. The oil palm fresh fruit bunches (FFB) was significantly reduced as early as Year 5 of harvest and continuously decreasing for four consecutive years until the Year 9 of harvest. Current results showed that even though the space between oil palm planting has increased to 18.3 m but the result remains the same. The reduction of oil palm FFB yield could be due to competition for light and nutrient between LTC rubber trees and oil palm. Yield produced from the LTC rubber trees would not be able to compensate losses from FFB yield. Therefore, this study concluded that LTC is not recommended for integration with oil palm in the double avenue oil palm planting system and also in standard triangular planting system.

EVALUATION OF CHEMICAL AND BIOLOGICAL TREATMENTS TO DEGRADE OIL PALM EMPTY FRUIT BUNCHES (Elaeis guineensis Jacq.) AND THEIR POTENTIAL USE

The exploitation of oil palm generates lignocellulosic wastes, also known as oil palm empty fruit bunches (EFB), which are difficult to biodegrade and are mainly composed of cellulose, hemicellulose and lignin. The presence of lignin impedes the use of the cellulose from this biomass in industrial processes. This study evaluated the effect of different chemical and biological treatments to remove the lignin present in the EFB from oil palm. The conventional kraft method, the soda-anthraquinone pulping process, microwaveassisted hydrogen peroxide degradation and the peroxy-monophosphoric acid method were the chemical treatments evaluated. Meanwhile, the four biological treatments were tested using different lignocellulolytic native fungi from the genera Xylaria, Psilocybe and Agrocybe. The results showed a greater reduction of the lignin content with an enrichment of cellulose using the kraft method and the soda-anthraquinone pulping process. For the biological treatments, the fungus Agrocybe sp. showed a selective delignification, exhibiting a remarkable potential for the utilisation of cellulose present in this type of waste. In contrast, the fungus Psilocybe sp. showed high non-selective fibre degradation, which could be used for bioremediation and composting systems.

EFFECT OF FLOW RATE AND CONCENTRATION OF CARBAMIDE ON THE REDUCING NOx EMISSIONS IN PALM BIODIESEL FUELED RESEARCH ENGINE

This study examines the consequences of concentration and flow rate of carbamide (urea) to reduce NOx emissions from a diesel engine propelled with biodiesel. Taguchi-Grey relational-based multi-response optimisation method was employed for test-plan. Results revealed that carbamide flow rate and its concentration have a positive impact on NOx emissions reduction in unmodified diesel engine. The obtained results are also justified with results attained from ANOVA. It is also confirmed that the results obtained in the experimentations are in line with the calculated values obtained Taguchi technique. Hence, the usage of carbamide in tailpipe is an effective way in reducing NOx emissions from diesel engine.

STABILITY OF PALM OLEIN WITH OR WITHOUT ANTIOXIDANTS DURING INDUSTRIAL CONTINUOUS DEEP-FAT FRYING OF WHEAT SNACKS

The stability of palm olein (POo) was evaluated during deep-fat frying of wheat pellet snacks in an industrial continuous frying system. Refined, bleached and deodourised POo free of antioxidants was employed, or with the addition of natural antioxidant (1000 ppm rosemary extract, RE) or with synthetic antioxidant (200 ppm tertbutylhydroquinone, TBHQ) at a rate of 975 kg hr -1. For comparison, frying with POo without antioxidants was carried out at a rate of 1800 kg hr -1. Several chemical indices in POo were evaluated. Also, sensory evaluation during storage of the final fried products was conducted. Addition of antioxidants did not have an effect on the free fatty acids during continuous frying (p>0.05). POo without antioxidants or added with RE had higher total polar compounds and p-anisidine values in comparison with POo with TBHQ. Measured indices were under recommended limits established by regulations, especially frying at 1800 kg hr -1, due to the rapid oil turnover in the continuous system. In the sensory evaluation during accelerated shelf-life test, no differences were found between snacks at different production rates and with or without addition of antioxidants (p>0.05). The POo showed high stability for continuous industrial frying of wheat snacks, producing foods with long shelf-life.

THE EFFECTS OF POLYGLYCEROL ESTERS ON PALM OLEIN FRACTIONATION

This study was undertaken to investigate the effects of adding 0.1% (w/w) – 0.9% (w/w) polyglycerol esters (PGE) on palm olein fractionation based on crystal nucleation, crystal growth and product quality aspects. Palm olein (IV 56) with PGE additives was subjected to crystallisation at a temperature of 13°C for 335 min. The crystal nucleation and its growth were monitored using focused beam reflectance measurement (FBRM) technique, during which the in-line changes of crystal size distribution during the process were analysed. The fractionated products were analysed for their iodine value (IV) and triacylglycerol (TAG) composition. The FBRM results showed that the PGE promoted crystal nucleation coupled with reduced formation of undesired large crystals during the crystallisation stage. The product analysis also showed that the PGE discouraged the formation of olein entrainment in the solid fraction but promoted the formation of 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content in the solid fraction. In addition, PGE also slowed the formation of 1-palmitoyl-2,3-dioleoy-glycerol (POO). The optimal dosage that offered best performance was 0.3% (w/w) PGE which gave the most medium sized crystals and best palm mid fraction product with lowest IV and highest content of POP. These results implied that PGE is a nucleation enhancing and crystal growth retarding additive in palm olein crystallisation at 0.3% (w/w) dosage.

EFFECT OF SUPERFATTING AGENTS ON SOAPS PROPERTIES

Advancements in soap technologies have improved the quality of life worldwide. Several new products have flooded the market encompassing laundry, beauty and dermatology. Over the years, palm oil (PO) has made an invaluable contribution in the global market. The use of palm stearin (POs) in soap making is well recognised due to its cost competitiveness. However, incorporation of POs in high amounts can cause cracking of soaps. The present studies focused upon the use of POs with palm kernel oil (PKO) in ratio of 60:40. This work is novel: i.e., pure distilled fatty acids (DFA) were incorporated as superfatting agents to upgrade soap properties. Neat soap was prepared by direct neutralisation and then superfatted with pure DFA of lauric, myristic, palmitic and stearic in proportions of 2%. Moisture content (MC) is an important controlling parameter during soap processing which effects the solubility and final texture of soaps. It is evidenced that superfatted (SF) soaps showed constancy in total fatty matter (TFM), free fatty acids (FFA), iodine value (IV) and peroxide value (PV) as the MC percentage declined with time. The stability of SF soaps persisted upon exposure at an elevated temperature of 100ºC for seven days. Also, the moisturising ability and foamability enhanced with the addition of superfatting agents. These soaps also exhibited improved resistance to cracking.

AN RFLP MARKER, CB75A, PREDICTS FOR PALMITIC ACID CONTENT ACROSS SELECTED OIL PALM GERMPLASM AND INTERSPECIFIC-HYBRIDS

Development of markers followed by construction of genetic linkage maps and quantitative trait locus (QTL) identification is a widely applied strategy towards marker assisted selection (MAS). In MPOB, researchers have reported that a restriction fragment length polymorphism (RFLP) marker, CB75A, generated from a OxG genetic linkage map showed significant correlation with QTL for iodine value (IV) and fatty acid composition (FAC) in oil palm. This indicated a potential use of the marker to assist breeding to develop genotypes with more liquid oil. The objective of this study is to validate the CB75A marker across various independent populations to gauge its utility for oil palm breeding. Palms were selected based on their palmitic acid composition, and classified as low, intermediate and high. Validation was carried out using RFLP technique on 18 Madagascar germplasm samples from Trial 0.240 at MPOB Kluang, 20 hybrids from Trial 0.236 at MPOB UKM and 60 backcross 1 (BC1) from Trial 0.336 at MPOB Kluang. Three bands were observed and assigned as A, B and C. The assay showed that the AA genotype is associated with low palmitic acid. In addition, significant segregation can only be observed in interspecific hybrid backcross samples. This inferred that although CB75A can predict FAC content to some extent, additional markers are required for a more precise prediction.

A TOOL FOR MONITORING Trichoderma AND Fusarium oxysporum f.sp. elaeidis OIL PALM INTERACTIONS, USING CONSTITUTIVE AND INDUCIBLE GREEN FLUORESCENT PROTEIN (GFP) AND RED FLUORESCENT PROTEIN (DsRED) REPORTER SYSTEM

Vascular wilt disease of oil palm is cause by Fusarium oxysporum f.sp elaeidis. The pathogen normally invades intact roots of palms or wounds and colonises the xylem vessels where it causes water stress, and hormonal imbalance result in severe yield loss and possible palm death. This study attempted to visualise the stages of colonisation and penetration into roots by Fusarium oxysporum f.sp elaeidis expressing Red Flourescent Protein (DsRed) in susceptible oil palm line and it’s interactions with Trichoderma TPP4 expressing Green Fluorescent Protein (GFP). Trichoderma TPP4 and Fusarium oxysporum f.sp elaeidis were successfully transformed using Agrobacterium tumefaciens-mediated transformation with both GFP and DsRed respectively using vectors pCAMDsRed and pCAMBgfp whereby this is the first report that Fusarium oxysporum f.sp elaeidis has been genetically modified. Analysis showed that early colonisation of Foe hyphae on the surface of secondary roots while colonisation by Trichoderma was observed at early stages after inoculation and became denser with time. Trichoderma TPP4 also was seen coiling around the Foe when inoculated together showing potential mycoparasitisic action.

OIL PALM ECONOMIC PERFORMANCE IN MALAYSIA AND R&D PROGRESS IN 2018 – Review Article

Staying resilient is probably an understatement for the oil palm industry during the period of 2018. Whilst other sectors such as the electrical and electronics and other manufactured products were experiencing a bullish growth, the export value of palm oil and palm oil-based products was on the decline. This is essentially due to the looming threat of a total ban of palm oil-based biodiesel by the European Union (EU) come 2021 and the higher production from palm oil plantations in Indonesia causing lower demand and oversupply worldwide. In view of the former, Malaysia’s export market scene is expected to change in the coming years. Malaysia has since identified several potential new markets in Africa, Central and South Asia. The negative campaigns relating to health, social or environment targeted at the oil palm are not new but the current action led by the West is clearly causing a major dent in the industry. As the most traded commodity, the industry as a whole is constantly subjected to scrutiny with environment being the current key issue warranting the imposition of checks and balances such as the Malaysian Sustainable Palm Oil (MSPO), Indonesian Sustainable Palm Oil (ISPO) and Rountable Sustainable Palm Oil (RSPO) certification. At the same time, every aspect of the research continues to progress further building on prior knowledge arising directly from working on the oil palm or through model systems. Their progress is briefly highlighted in this review. Clearly the main theme of the research from upstream to midstream and finally downstream are basically aligned with and directed towards achieving the Sustainable Development Goals (SDG) by 2030. This would consequently help further improve the perception of oil palm/palm oil in general.