Category Archives: 2008 Special Issue Apr

Application of spectroscopic methods for the automation of oil palm culture

Oil palm tissue culture offers a potentially practical route to clonal propagation of high yielding palms. However, current tissue culture methods are laborious and costly, and the performance of the cultures can be difficult to describe quantitatively. Computer control of bioreactor processes increases reproducibility and permits quantitative description of the growth of oil palm cultures. Even so, there remain unmet needs in the areas of online metabolite measurement and of automation of the tissue culture process. In this work, we apply Raman spectroscopy for non-destructive off-line quantitation of sucrose, glucose, fructose, nitrate, potassium phosphate and magnesium sulphate metabolites in oil palm bioreactor culture supernatants. We also explore the feasibility of using fluorescence to discriminate between different morphotypes of oil palm calli. Finally, we report the use of flow cytometry to sort oil palm suspension cultures on the basis of size; selected samples were deposited into separate wells in a microplate with one callus particle per well. The technologies described in this article contribute to the development of automated methods for moving and positioning oil palm cells, and for online measurement of metabolites in oil palm bioreactor supernatant.

Isolation and characterization of a putative serine/threonine kinase expressed during oil palm tissue culture

One of the key processes hindering the efficiency of oil palm tissue culture is its poor embryogenesis rate and therefore efforts to pursue improvements in this area remain essential. EgPK1, a homolog of the mammalian STK16, was isolated from oil palm embryogenic suspension cultures through cold plaque screening. Upregulation of EgPK1 transcripts was observed in embryogenic calli relative to its non-embryogenic counterpart. Quantitative PCR also demonstrated similar results. A 5’RACE fragment was found to be different from the original EgPK1 sequence, with the variation unique to the first 115 nucleotides at the 5’region, suggesting alternative splicing events. Quantitative PCR analyses suggested that expression profiles of the variants across different developmental stages were similar to each other. As hypothesized in the mammalian systems, EgPK1 may serve a similar role in disruption of the extracellular matrix surrounding the proembryogenic masses during somatic embryogenesis.

Effect of agitation and aeration on yield optimization of oil palm suspension culture

Oil palm is an oil producing tropical plant. While it is the highest yielding oil producing plant, it takes three years to yield. Therefore there is an urgent need for controlled production for oil palm suspension cells in a bioreactor to support the clonal propagation of elite planting materials. The propagation of the cells is affected by various culture conditions. In this study, we examined the effect of two operating parameters – agitation rate (80 to 335 rpm) and aeration (10% to 80% dissolved oxygen) – on the proliferation of suspension cells. The sugar consumption, production of biomass, amino acids and organic acids were monitored. At 43% dissolved oxygen, two of the agitation rates (120 rpm and 225 rpm) resulted in more than 200% increase in biomass compared to the initial inoculum. This study indicates that the bioreactor operating parameters between 120-300 rpm and 20%-80% dissolved oxygen are acceptable for the culture of oil palm cells.

Functional annotation of oil palm genes using an automated bioinformatics approach

Recent advances in DNA sequencing technologies have led to a tremendous increase in the amount of sequence information available in public databases. To address the need for automated methods of assigning a putative function to each sequence, we have developed bioinformatics tools that can be run on a desktop computer and save significant time and effort. Elaeis guineensis and Elaeis oleifera sequences were downloaded from PalmGenes and GenBank, and duplicate entries were eliminated by pairwise BLAST searches, resulting in a collection of unique oil palm sequences which we call the UniPalm dataset. We applied the CAPASA (Consensus Annotation by Phrase Anchored Sequence Alignment) software and automatically assigned functions to 5600 oil palm sequences in less than 8 hr. CAPASA mimics the human decision-making process by factoring in the degree of homology, taxonomic relationship and informational value when choosing a name. In addition, we applied COGsensus to place the UniPalm sequences into COG (Clusters of Orthologous Groups of genes) categories, and compared these results to a COGsensus analysis of the rice genome. COG classification is a homology-based method for distinguishing gene sets, particularly with regard to closely related genes found in different organisms. Our results indicate that the diversity of COG groups are well represented in the UniPalm set.

Multiplication of oil palm liquid cultures in bioreactors

Bioreactor systems can provide quantitative data on oil palm liquid cultures since control of the environmental variables is maintained. Oil palm liquid cultures were multiplied in three bioreactors – B-Braun, Biotron and Sixfors. The cultures showed good proliferation with about 5- to 12-fold weight increment after about 60 days in the B-Braun bioreactor. Increments in fresh weight of four-to five-fold were obtained from the Biotron bioreactor. In the Sixfors system, the increment in cell fresh weight was about three-fold. The production and productivity of cultures multiplied in the bioreactors varied from one clone to another. This study has provided a better understanding of oil palm liquid cultures with regards to their growth in different bioreactors and their potential for commercial applications.

Identification of genes expressed in the embyryoid tissue of oil palm (Elaeis guineensis Jacq.) tissue culture via expressed sequence tag analysis

In vitro propagation is the only means of vegetatively propagating oil palm. Despite this, understanding of the molecular basis of oil palm tissue culture, especially embryogenesis, remains poor. Therefore, this study used expressed sequence tags (ESTs) to investigate the genes expressed during embryogenesis of oil palm tissue culture. Total RNA was extracted from oil palm embryoid tissue. Spectrophotometry and agarose gel electrophoresis indicated that the RNA molecules were intact and suitable for complementary DNA (cDNA) library construction. Two cDNA libraries were constructed using mRNAs from oil palm embryoid tissue. The two cDNA libraries were Embryoid N (EN) and Embryoid O (EO). The primary titre for the EN cDNA library was 4.52 x 105 pfu, while the primary titre for the EO cDNA library was 1.20 x 106 pfu. The average insert sizes of the EN and EO cDNA libraries were 1.0 kb and 0.8 kb respectively. Subsequently, the EO cDNA library was chosen for generation of ESTs. Plasmid extraction was carried out for random recombinant clones picked from the cDNA library, and a total of 6535 recombinant clones were found suitable for DNA sequencing. A total of 5247 ESTs with PHRED score >20 and sequence length < 100 bp were generated. Cluster analysis generated 3545 unique transcripts with 2692 singletons and 853 consensus sequences. Similarity search showed that 70% (2484/3545) of the unique transcripts had significant similarity (E value <10-10) to sequences in GenBank. Gene function classification showed that the genes highly expressed are those involved in metabolism (16%), protein destination, modification and storage (10%), defence, development, ageing, disease and stress (9%). Among the genes identified are those that may potentially be involved in embryogenesis, such as the lipid transfer protein homolog (WBP1A), somatic embryogenesis receptor kinase 1 (SERK1) and defensin EGAD1. The results showed that the EST approach is an effective strategy in gene discovery and capable of generating important and useful information for gene expression studies in oil palm tissue culture.