An Examination of Oil Palm Flowering: Molecular Biology and Anatomical Analysis

Flowering is an important part of oil palm crop production The ability to control flowering will be economically beneficial to the oil palm industry Before control can be exerted, factors that influence flowering must be studied at the level of physiology, biochemistry and molecular biology The morphological aspects of oil palm flower development were examined by light and electron microscopy. In doing so, oil palm flower development was able to be partially staged for future reference to determine the expression pattern of flower-predominant genes. Protein analysis was carried out to identify unique proteins that are expressed in the vegetative meristem or the floral meristem. Three proteins were identified, the presence or absence of which could be associated with the conversion of a vegetative meristem into an inflorescence meristem. Histological studies made on the flowers produced by the inflorescence meristem revealed that these flowers were not true flowers. These flower-like structures were not found to have any reproductive structures and only contained bracts. This reflected that in vitro, under high cytokinin growth condition, the conversion of inflorescence meristem to a floral meristem is disrupted. Flower-predominant clones were isolated from an oil palm male flower cDNA library by a differential screening procedure. Four out of 10 floralpredominant clones were characterized. Clone 68 has homology to cytochrome P450 while clone 65, 70 and 72 do not have any significant homology with any known functional genes but their characteristics showed that clones 65 and 70 could be a glycoprotein and clone 72, a protein kinase. However, the functions of these genes in oil palm flowering are still unconfirmed

Isolation and characterization of EgGST, a glutathione S-transferase protein transcript in oil palm (Elaeis guineensis Jacq.)

The formation of callus and somatic embryos remains one of the major bottlenecks in oil palm tissue culture. Unlike other crops, oil palm tissue culture is a very slow process. In the present study, EgGST (GenBank accession no. AIC33066.1), an oil palm gene coding for a putative glutathione S-transferase protein, has been characterized molecularly. The full length cDNA sequence of EgGST isolated from oil palm cultured leaf explants at the 6th week is 1002 bp in length, with an Open Reading Frame (ORF) of 651 bp. The deduced EgGST encodes a 216-amino-acid protein with a predicted molecular mass of 23.68 kD and a pI value of 6.16. Its protein sequence shares 63% identity with the glutathione s-transferase gstf2 from Oryza sativa Indica Group (GenBank accession no. ABR25713.1) and contains thioredoxin fold and chloride channel domain. Real-time RT-PCR results showed that the EgGST transcript was differentially expressed across a time series of fortnightly-cultured leaf explants and had a higher transcript levels in nodular callus (NC) compared to friable callus (FC) for oil palm ortet of clone 4178. EgGST was also found to be preferentially expressed in all tissue culture derived materials except for oil palm cell suspension culture (CSC), whereas there were almost negligible expressions in all the non-tissue culture derived materials, except for root. Hence, it can be suggested that EgGST transcript may possibly be regulated differently at different stages of tissue culture and various tissues. Interestingly, EgGST also displayed a tissue-specific expression pattern via RNA in situ hybridization. To our knowledge, this is the first reported study on the analysis of the localization of target mRNA transcript of EgGST in different oil palm tissues. We postulated that EgGST might play significant roles at different stages of oil palm callogenesis, and could potentially be a candidate marker for oil palm callogenesis

Recycling of oil palm empty fruit bunch as potential carrier for biofertilizer formulation

The palm oil industry generates large amount of biomass waste such as oil palm empty fruit bunch (EFB) and palm oil mill effluent (POME). This biomass would be typically recycled to produce EFB compost that is a valuable agricultural input. This study was conducted to evaluate the suitability of using EFB compost as carrier for nitrogen fixing bacteria (NFB) and phosphate solubilizing bacteria (PSB). Mixture (50-60% moisture) between EFB (shredded short fibres) and POME (anaerobic pond) were added with Effective Microorganisms (EM) to accelerate the composting process. The EFB compost reached thermophilic phase after 4-6 weeks with consistent temperature between 50- 60°C. After 7 weeks of composting, EFB compost reached the mesophilic phase with continuous reduction of temperature to 35-40°C at week 8. The maturity of the compost is supported from the reduction of the C/N ratio from 36 (initial) to 20 (after 6 weeks). Mature EFB compost was sun-dried and ground into fine particle size (1 mm) prior to be used as carrier. Dried EFB compost carrier has the following characteristics (dry weight); average pH at 7.5, C/N ratio - 13.5, moisture - 17.4%, organic matter – 74.5%, total N - 3.06%, P - 0.37%, K - 4.74%, Ca - 3.32% and Mg - 0.79%. The inoculation of NFB and PSB into the EFB compost carrier from single cultures of Serratia marcescens and Enterobacter cloacae showed high viable cell count at 4.05 × 109 CFU/g and 2.75 × 108 CFU/g respectively at day three after inoculation. Meanwhile, the mixed culture of Burkholderia cenocepacia with Serratia marcescens showed 2.45 × 108 CFU/g and 4.31 × 109 CFU/g respectively. This clearly indicates the potential of using EFB as a useful alternative for bacterial immobilization prior to application in the oil palm industries

Analyses using genomic and transcriptomic data of mads-box genes in Elaeis guineensis

MADS-box genes encode a group of transcription factor family, well known as key regulators of plant vegetative and development processes, including flowering. Here, we aimed at analysing MADS-box genes in African oil palm (Elaeis guineensis) genome, the pre-dominant source of worldwide production of vegetable oils. A total of 209 potential MADSbox genes are identified in the published 1.8 Gb E. guineensis draft genome. A de novo assembly of RNA-seq data from inflorescence tissues was constructed using Trinity software. The analysed transcriptome data has validated 36 full-length genes, inclusive of seven transcripts that were previously annotated to encode unknown proteins. Of the 36 genes discovered, 21 genes were characterised as Type I MADS-box genes and phylogenetic analysis using maximum likelihood approach further classified them into three sub-groups of Mα, Mβ and Mγ. Based on in silico analyses, we have successfully identified one gene annotated as unknown protein to contain a domain of “MADS-box transcription factor”. Its differential expression data, comparison between normal and mantled inflorescence of oil palm, suggested the involvement of the gene in the mantling related process. The findings contribute to updated oil palm genome information which may potentially lead to future understanding of the association of MADS-box genes in mantled oil palm. This may also lead to a resource for biomarker discovery

Recycling of superfine resolution agarose gel

Genetic markers are now routinely used in a wide range of applications, from forensic DNA analysis to marker-assisted plant and animal breeding. The usual practice in such work is to extract the DNA, prime the markers of interest, and sift them out by electrically driving them through an appropriate matrix, usually a gel. The gels, made from polyacrylamide or agarose, are of high cost, limiting their greater applications in molecular marker work, especially in developing countries where such technology has great potential. Trials using superfine resolution (SFR) agarose for SSR marker screening showed that it is capable of resolving SSR loci and can be reused up to 14 times, thus greatly reducing the cost of each gel run. Furthermore, for certain applications, low concentrations of agarose sufficed and switching to lithium borate buffer, instead of the conventional Tris-borate-ethylenediaminetetraacetic acid buffer, will further save time and cost. The 2.5% gel was prepared following the Agarose SFR™ manual by adding 2.5 g agarose powder into 100 mL 1X lithium borate buffer in a 250-mL flask with rapid stirring. Two midigels (105 x 83 mm, 17 wells) or 4 minigels (50 x 83 mm, 8 wells), 4 mm thickness can be prepared from 100 mL gel solution. A total of 1680 PCR products amplified using 140 SSR markers from oil palm DNA samples were tested in this study using SFR recycled gel. As average, the gel can be recycled 8 times with good resolution, but can be recycled up to 14 times before the resolutions get blurred

Differentially expressed transcripts related to height in oil palm

A subtractive cDNA library was constructed using MPOB Planting Series 1 (PS1) population to isolate differentially expressed transcripts associated with height increment in oil palm. After differential screening, 98 clones were identified to be potentially positive with cDNA inserts ranging from 250 to 1000 bp. A total of 123 sequences generated and low quality sequences ≤20 were eliminated using Phred program to assess the sequence quality and determine the accurate consensus sequence. Contig assembly by CAP3 program generated 51 contigs and four singletons (55 unigenes). Blast search showed 49 unique sequences had significant match to various plant species in the GeneBank database with E-value ≤1e-5, and four sequences showed no significant similarity. Gene ontology analysis output from Blast2 GO program revealed that the sequences encoding for auxin responsive protein, circadian clock-associated protein1, zinc-finger protein and basic leucine zipper protein were potentially associated to dwarfism. The genes were identified based on their putative functions in regulating height in plants, particularly in growth hormone biosynthesis such as auxin (Aux/IAA), gibberellins (GA) and brassinosteroids (BR)

Isolation and characterization of differentially expressed transcripts from the suspension cells of oil palm (Elaeis guineensis Jacq.) in response to different concentration of auxins

Oil palm suspension cultures were initiated by transferring the gel-like friable embryogenic tissue onto liquid medium supplemented with auxins. In this study, transcripts that were differentially expressed in oil palm suspension cells cultured at different auxin concentrations were examined using suppression subtractive hybridization. Total RNA was first isolated from oil palm suspension cells proliferated in liquid medium with different hormone concentrations for 6 months. Four different hormone combinations: T1 (0.1 mg/l 2,4-D and 1.0 mg/l NAA), T2 (0.4 mg/l 2,4-D and 1.0 mg/l NAA), T3 (1.0 mg/l NAA), and T4 (0.4 mg/l 2,4-D) were used for the treatments. The first and second subtractions were performed using samples T1 and T2 in forward and reverse order. The other two subtractions were forward and reverse subtractions of T3 and T4, respectively. Reverse northern analyses showed that 14.13% of these clones were preferentially expressed in T1, 13.70% in T2, 14.75% in T3, and 15.70% in T4. Among the 294 cDNA clones that were sequenced, 61 contigs (assembled from 165 sequences) and 129 singletons were obtained. Among the 61 contigs, 10 contigs consist of sequences from treatment T1, 8 contigs were from treatment T2, 10 contigs were contains sequences of treatment T3 and 13 contigs contains sequences of treatment T4. Northern analyses of five transcripts that were shown to be differentially expressed in the oil palm suspension cells by reverse northern analysis revealed that transcripts 16A1 (a putative lignostilbene-α,β-dioxygenase, EgLSD) and 16H12 (a putative ethylene responsive 6, EgER6) were differentially expressed in oil palm suspension cells treated with different levels of auxin