Optimization of Parameters Involved in the Transformation of Oil Palm Using the Biolistic Method

Physical and biological parameters affecting DNA delivery into oil palm embryogenic calli using the biolistic device have been optimized. The physical parameters tested were : helium pressure, distance from rupture disc to the macro carrier, distance from macro carrier to the stopping plate, distance from stopping plate to the target tissue, vacuum pressure, number of bombardments, particle types and sizes, and the effect of calcium chloride and spermidine on microcarrier-DNA binding. The optimized biological parameters were: explant types with gold micro carrier, explant types with tungsten, duration of callus culture in fresh medium prior to bombardment, duration between bombardment and GUS staining, genotype, immature embryo preculture duration, DNA concentration, osmoticum type and concentration and osmoticum treatment duration before and after bombardment. Independent experiments were carried out to study the effects of each parameter and its variables on transient expression. Two days after bombardment, the tissues were stained with GUS assay buffer for 16-20 hours at 37°C and the blue spots counted under a binocular microscope. All the variables used in these experiments were found to be significantly different except for vacuum pressure, bombardment number and genotype. The efficiency of GUS gene expression was measured in embryogenic calli and young leaves of mature and seedling palms using five constructs carrying different promoters : Emu; Ubil; Actl, 35S and Adhl were evaluated to identify the most suitable promoter for use in oil palm. The GUS gene expression from the different promoters was assayed histochemically and fluorometrically from a total of 200 plates of target tissues in eight independent experiments. Significant effects on transient GUS gene expression were demonstrated by each of the different promoters tested. The effectiveness of kanamycin; geneticin (G-418); neomycin, hygromycin and basta as selection agents to inhibit growth of oil palm embryogenic calli was evaluated. Embryogenic calli were separately exposed to all these selection agents at different concentrations ranging from 1 to 2000 mg/l for a period of one month. This was done in two replicates and repeated twice to ensure reproducibility of the selection system. Of the five compounds tested, hygromycin and basta were found to be most suitable as selection agents for oil palm as they can stop the growth of embryogenic calli at lower concentrations.Bombarded embryogenic calli were exposed to 40 or 80mg/l of selective agents after 1 or 3 weeks. It was found that there were no significant differences in the number of resistant embryogenic calli produced per plate when selected at different concentrations and time. The presence of transgenes in the resistant embryogenic calli was confirmed by PCR and Southern analysis. Transgenic embryogenic calli were later regenerated into whole plants and their transgenic status verified by PCR and Southern analysis. Problems faced during the study and their solutions are also discussed. As oil palm has a long breeding cycle, inheritance of transgenes cannot be demonstrated within the period of this study. Therefore, rice, a model crop for monocot transformation, was also used for transformation experiments. Calli derived from immature embryos were bombarded and were selected on hygromycin. Resistant calli isolated were regenerated into whole plants. Two transgenic lines were obtained. T1 and T2 from one of the clones were also produced and analysed. Integration and inheritance of the transgenes were followed by phenotypic and genotypic analysis

Transgenic plants producing polyhydroxyalkanoates

Currently, the polyhydroxybutyrate (PHB) copolymer, polyhydroxy-co-valerate (PHBV) is being produced commercially using a two-stage glucose / propionate fed batch fermentation process using Ralstonia eutropha. The economics of the manufacturing process are still a major barrier to the widespread use of polyhydroxyalkanoates (PHAs) and intensive efforts are being made to reduce the cost of production by means of bioprocess design and metabolic engineering of production strains. However, despite these improvements, the production costs are still high compared to petroleum-derived commodity plastics. An alternative strategy for lowering production costs that has been proposed is to develop transgenic plants that produce PHAs. This strategy is considerably cheaper because the PHAs production from plants does not require expensive fermentation equipment and processing facilities

Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L)

Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1-1 mannose without sucrose. Transgenic plants were verified using PCR analysis

Construction of PHB and PHBV transformation vectors for bioplastics production in oil palm

The construction of transformation vectors carrying bioplastic biosynthetic genes driven by constitutive and oil palm mesocarp-specific promoters was completed. Four planned transformation vectors were produced. The poly-3-hydroxybutyrate (PHB) producing constructs carried the phbA, phbB mid phbC genes, while the polyhyroxybutyrate-co-valerate (PHBV) producing constructs carried the bktB, phbB, phbC and tdcB genes. Each of these genes was fused with the transit peptide (Tp) of the oil palm acyl-carrier-protein (ACP) for targeting into the plastids of plant cells. All vectors carry the phosphinothricin acetyltransferase gene (bar) driven by an ubiquitin promoter as plant selectable marker. The matrix attachment region from tobacco (RB7MAR) was also included for stabilization of the transgene expression and to minimize the gene silencing due to positional effects. All constructs were verified by restriction analysis, polymerase chain reaction (PCR) and DNA sequencing

Factors affecting delivery and transient expression of β-glucuronidase gene in Dendrobium Sonia protocorm-like-body

The effect of the biolistic device parameters and other factors affecting delivery and expression of uidA gene in Dendrobium Sonia was investigated. Three week old protocorm like body (PLB) were mombarded with gold microparticles coated with pAHC25 plasmid harbouring the uidA gene which encodes β-glucuronidase. The factors investigated were the helium pressure, target distance, macrocarrier flight distance to stopping screen, distance from stopping screen to target tissues, vacuum pressure, gold microparticles size, spermidine and calcium on DNA precipitation, and the number of bombardments. Two days after bombardment, the PLB were subjected to histochemical GUS assay, and transient GUS activity was recorded as blue spots using a Leica streomicroscope. All the factors tested showed significant effects (p<0.05) on the delivery of DNA and expression of the uidA gene in Dendrobium PLB except for calcium. Surviving PLBs were able to grow and regenerate normally into plantlets

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

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 Duncan's 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 μg of gold microcarriers coated with 1.5 μg 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

Construction of PHB and PHBV multiple-gene vectors driven by an oil palm leaf-specific promoter

One of the targets in oil palm genetic engineering programme is the production of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) in the oil palm leaf tissues. Production of PHB requires the use of phbA (β-ketothiolase type A), phbB (acetoacetyl-CoA reductase) and phbC (PHB synthase) genes of Ralstonia eutropha, whereas bktB (β-ketothiolase type B), phbB, phbC genes of R. eutropha and tdcB (threonine dehydratase) gene of Escherichia coli were used for PHBV production. Each of these genes was fused with a transit peptide (Tp) of oil palm acyl-carrier-protein (ACP) gene, driven by an oil palm leaf-specific promoter (LSP1) to genetically engineer the PHB/PHBV pathway to the plastids of the leaf tissues. In total, four transformation vectors, designated pLSP15 (PHB) and pLSP20 (PHBV), and pLSP13 (PHB) and pLSP23 (PHBV), were constructed for transformation in Arabidopsis thaliana and oil palm, respectively. The phosphinothricin acetyltransferase gene (bar) driven by CaMV35S promoter in pLSP15 and pLSP20, and ubiquitin promoter in pLSP13 and pLSP23 were used as the plant selectable markers. Matrix attachment region of tobacco (RB7MAR) was also included in the vectors to stabilize the transgene expression and to minimize silencing due to positional effect. Restriction digestion, PCR amplification and/or sequencing were carried out to ensure sequence integrity and orientation

Identification of oil palm (elaeis guineensis) spear leaf metabolites using mass spectrometry and neutral loss analysis.

Plant metabolites characterisation is an arduous challenge due to their structural diversity as a result of complicated biosynthetic pathways. These metabolites are not only important for metabolic events description but are also harnessed as valuable nutraceuticals. The detection and description of important plant tissue metabolome such as oil palm spear leaf can be carried out using a broad-range, non-targeted analytical tool such as mass spectrometry (MS). Identification of oil palm spear leaf constituents such as carboxyl group- and sugar- containing metabolites was facilitated by tandem mass spectrometry (MS/ MS) with neutral loss information of 44 and 162 amu. A total of 13 metabolites ranging from carboxylic acids, catechins, phenolic acid glycosides and a stilbenoid were characterised in this manner and the results presented here demonstrated the usefulness of MS in characterising metabolites in a complex sample such as oil palm spear leaf tissue

Tissue specific promoters: the importance and potential applications for genetic engineering in oil palm

Oil palm is the most prolific oil crop in the world with a productive life span of 20-30 years and this perenniality bestows significant advantages over other oil crops. However, the industry still faces a number of challenges and to ensure its future sustainability, efforts must be made to diversify applications to increase its economic value. Amongst potential strategies include the use of genetic engineering approaches to fulfil the needs. To ensure that expression of transgenes for the production of genetically engineered products is directed to targeted tissue(s), promoter sequences that are responsible to direct the expression of desired genes have been identified. In this review we discuss the isolation and characterisation of oil palm tissue-specific promoters from mesocarp and kernel, an inducible tissue-specific promoter from roots, and the utility of constitutive promoters. The tissue-specific and constitutive functions of these promoters were confirmed through transient expression studies in oil palm and some of the isolated promoters were further characterised using Arabidopsis as a model system. We hope that these promoters can potentially be utilised to improve oil yield and quality, to fine tune the agronomic traits, and to generate high value-added products for the oil palm