Journal of Oil Palm Research (Special Issue - April 2008), p. 51-63 ELYANA Md Shariff * ; LESLIE LOW Eng Ti * ; HALIMAH Alias ** ; CHEAH Suan Choo * ; SINGH, Rajinder*
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.KEYWORDS:
* Malaysian Palm Oil Board, P. O. Box 10620, 50720 Kuala Lumpur, Malaysia.
** Malaysian Genome Institute,
UKM-MTDC Smart Technology Centre,
Universiti Kebangsaan Malaysia,
43600 UKM Bangi, Selangor, Malaysia.
Journal of Oil Palm Research Special Issue on Malaysia-MIT Biotechnology Partnership Programme: Volume 1-Oil Palm Tissue Culture