Norihan M. Saleh

Physical parameters affecting transient GUS gene expression in oil palm (Elaeis guineensis Jacq.) using the biolistic device.

Abstract Physical parameters for DNA delivery into oil palm embryogenic calli using the biolistic device have been successfully established. Helium pressures, distance from rupture disc to macrocarrier, distance from macrocarrier to stopping plate, distance from stopping plate to target tissue, vacuum pressures, number of bombardments, particle types and sizes and the effect of calcium chloride and spermidine on microcarrier-DNA binding have been optimizied. Embryogenic calli were bombarded with gold or tungsten particles coated with pEmuGN plasmid containing β-glucuronidase gene ( uidA ) driven by an Emu promoter. A completely randomized design was used to bombard a total of 200 plates (40 parameters × 5 replications) which were then incubated in a growth chamber at 28 °C. Two days after bombardment, the tissues were stained with GUS assay buffer for 16–20 h at 37 °C and the blue spots were counted under a binocular microscope. Analysis of variance (ANOVA) and Duncans new multiple range test were used to analyze data from each experiment. All the variables used in this experiment were significantly different except for vacuum pressures and bombardment numbers.

Evaluation of five promoters for use in transformation of oil palm (Elaeis guineensis Jacq.).

The efficiency of GUS (β-Glucuronidase) gene expression in embryogenic callus and young leaflets of mature and seedling palm after microprojectile bombardment with five constructs (pEmuGN, pAHC25, pAct1-F4, pGH24 and pBARGUS) was evaluated to identify the most suitable promoter(s) to use in transformation attempts in oil palm. Expression of the GUS gene driven by theEmu, Ubi1, Act1 35S orAdh1 was assayed, both histochemically and fluorometrically, from a total of 200 plates of tissues in eight independent experiments two days after bombardment. A completely randomized experimental design was used for each experiment, and the data analysed by ANOVA and Duncans Multiple Range Test. The expression level of GUS driven by theEmu orUbi1 promoters was significantly higher than that of the Act], 35S and Adhl promoters in many experiments, and that of theAdhl was significantly lower than those of the other four promoters. Both histochemical and fluorometric data indicate that in embryogenic callus, the expression of theEmu promoter was higher than that of theUbi1 whereas in young leaflets from mature palm the Ubi1 expression was stronger. The performances of the five promoters were also tested in tobacco callus using a fluorometric GUS assay. The activity of the 35S promoter was highest, and significantly different from that of all the other promoters except theEmu, and that of theAct1 promoter was lowest. These results indicate that either theUbil orEmu promoter should facilitate the expression of desired genes in oil palm and aid in development of an efficient stable transformation system.

Utilisation of enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR to fingerprint the genomes of Bifidobacterium isolates and other probiotic bacteria

Enterobacterial repetitive intergenic consensus based on PCR (ERIC-PCR) was used to generate DNA fingerprints for bifidobacteria and other probiotic bacteria. Two primers (ERIC 1R and ERIC 2) used in ERIC-PCR revealed that all of the probiotic bacteria tested possess enterobacterial repetitive intergenic consensus sequences with the PCR products ranging from 250 bp to 5000 bp. The bacterial strains can be differentiated by comparing fingerprint patterns. The dendrogram of the fingerprints revealed that most of the bifidobacterial wild type strains fell into one cluster at similarity level of approximately 79%.

Biological parameters affecting transient GUS gene expression in oil palm (Elaeis guineensis Jacq.) embryogenic calli via microprojectile bombardment

Abstract Biological parameters affecting microprojectile bombardment delivery of DNA into oil palm embryogenic calli were optimized by monitoring transient GUS gene expression. The parameters optimized were the following: explant type using gold and tungsten microcarrier, bombardment preculture, time between bombardment and GUS staining, genotype, immature embryo preculture, DNA concentration, osmoticum type and concentration, and osmoticum treatment duration before and after bombardment. An independent experiment was carried out to study the effect of each parameter and its variables on transient GUS expression. ANOVA was carried out for each experiment using completely randomized design and the treatment means were compared using Duncans Multiple Range Test. The highest transient GUS expression was observed 2 days post-bombardment using embryogenic calli derived from immature embryos. Bombardment was carried out using 300 μ g of gold microcarriers coated with 1.5 μ g of DNA 24 h after transfer to fresh medium. GUS expression could be further enhanced when calli were transferred to medium containing osmoticum (0.4 M mannitol) 2 h prior to bombardment. Highly significant differences between the variables were observed for all the parameters studied except genotype.

Cloning and sequence analysis of bile salt hydrolase (bsh) gene from Bifidobacterium longum

A pair of PCR primers for the rapid detection of bile salt hydrolase (bsh) gene from Bifidobacterium longum BB536 has been synthesised and have revealed the bsh gene of approx 970 bp in Bifidobacterium longum BB 536 but not in other species of bacteria tested. The bsh gene was cloned and sequenced showing a high similarity to bsh gene previously published. The resulting nucleotide sequence encodes a predicted protein of 317 amino acids, Mw = 35 kDa.

Analysis of expressed sequence tags derived from inflorescence shoot of Tectona grandis (teak)

5pfu/ml (primary) and 4.5 x 10 9 pfu/ml (amplified). EST generation and analysis were performed using the cDNA library where a total of 1384 plaques were randomly picked and their inserts PCR-amplified using T3 and T7 universal primers. Only 1125 plaques generated single amplified fragments, each which were purified and sequenced using the SK universal primer. The generated raw 5’ ESTs were filtered and clustered. A total of 674 nonredundants (69 consensus sequences and 605 singletons) were generated and their identities searched through BLASTX. Of the 674 nonredundants, 107 of them (15.9%) showed no hits or no identity. All the 567 nonredundants identified through BLASTX were categorized into their functional categories and were further analysed using InterProScan to detect their protein signatures and to assign their GO numbers. From all the sequences analysed, only 186 (32.8%) sequences were given the GO numbers and grouped into the three GO main categories namely biological process, cellular component and molecular function. Several important ESTs were highlighted based on their functional categories. There were five sequences found to be related to flowering and light induction.