Ghulam Kadir Ahmad Parveez

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.

Transgenic oil palm: production and projection.

Oil palm is an important economic crop for Malaysia. Genetic engineering could be applied to produce transgenic oil palms with high value-added fatty acids and novel products to ensure the sustainability of the palm oil industry. Establishment of a reliable transformation and regeneration system is essential for genetic engineering. Biolistic was initially chosen as the method for oil palm transformation as it has been the most successful method for monocotyledons to date. Optimization of physical and biological parameters, including testing of promoters and selective agents, was carried out as a prerequisite for stable transformation. This has resulted in the successful transfer of reporter genes into oil palm and the regeneration of transgenic oil palm, thus making it possible to improve the oil palm through genetic engineering. Besides application of the Biolistics method, studies on transformation mediated by Agrobacterium and utilization of the green fluorescent protein gene as a selectable marker gene have been initiated. Upon the development of a reliable transformation system, a number of useful targets are being projected for oil palm improvement. Among these targets are high-oleate and high-stearate oils, and the production of industrial feedstock such as biodegradable plastics. The efforts in oil palm genetic engineering are thus not targeted as commodity palm oil. Due to the long life cycle of the palm and the time taken to regenerate plants in tissue culture, it is envisaged that commercial planting of transgenic palms will not occur any earlier than the year 2020.

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.

Efficient Transformation of Oil Palm Protoplasts by PEG-Mediated Transfection and DNA Microinjection

Background Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. Methodology/Principal Findings We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. Conclusions/Significance We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants.

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 (beta-ketothiolase type A), phbB (acetoacetyl-CoA reductase) and phbC (PHB synthase) genes of Ralstonia eutropha, whereas bktB (beta-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.

Biotechnology of oil palm: strategies towards manipulation of lipid content and composition

Oil palm is a major economic crop for Malaysia. The major challenges faced by the industry are labor shortage, availability of arable land and unstable commodity price. This has caused the industry to diversify its applications into higher value products besides increasing its yield. While conventional breeding has its limitations, biotechnology was identified as one of the tools for overcoming the above challenges. Research on biotechnology of oil palm began more than two decades ago leveraging a multidisciplinary approach involving biochemical studies, gene and promoter isolation, transformation vector construction and finally genetic transformation to produce the targeted products. The main target of oil palm biotechnology research is to increase oleic acid in the mesocarp. Other targets are stearic acid, palmitoleic acid, ricinoleic acid, lycopene (carotenoid) and biodegradable plastics. Significant achievements were reported for the biochemical studies, isolation of useful oil palm genes and characterization of important promoters. A large number of transformation constructs for various targeted products were successfully produced using the isolated oil palm genes and promoters. Finally transformation of these constructs into oil palm embryogenic calli was carried out while the regeneration of transgenic oil palm harboring the useful genes is in progress.

Production of polyhydroxybutyrate in oil palm (Elaeis guineensis Jacq.) mediated by microprojectile bombardment of PHB biosynthesis genes into embryogenic calli

Biodegradable plastics, mainly polyhydroxybutyrate (PHB), which are traditionally produced by bacterial cells, have been produced in the cells of more than 15 plant species. Since the production of biodegradable plastics and the synthesis of oil in plants share the same substrate, acetyl-coenzyme A (acetyl-CoA), producing PHB in oil bearing crops, such as oil palm, will be advantageous. In this study, three bacterial genes, bktB, phaB, and phaC, which are required for the synthesis of PHB and selectable marker gene, bar, for herbicide Basta resistant, were transformed into embryogenic calli. A number of transformed embryogenic lines resistant to herbicide Basta were obtained and were later regenerated to produce few hundred plantlets. Molecular analyses, including polymerase chain reaction (PCR), Southern blot, and real-time PCR have demonstrated stable integration and expression of the transgenes in the oil palm genome. HPLC and Nile blue A staining analyses confirmed the synthesis of PHB in some of the plantlets.

Regeneration of viable oil palm plants from protoplasts by optimizing media components, growth regulators and cultivation procedures

Oil palm protoplasts are suitable as a starting material for the production of oil palm plants with new traits using approaches such as somatic hybridization, but attempts to regenerate viable plants from protoplasts have failed thus far. Here we demonstrate, for the first time, the regeneration of viable plants from protoplasts isolated from cell suspension cultures. We achieved a protoplast yield of 1.14×10(6) per gram fresh weight with a viability of 82% by incubating the callus in a digestion solution comprising 2% cellulase, 1% pectinase, 0.5% cellulase onuzuka R10, 0.1% pectolyase Y23, 3% KCl, 0.5% CaCl2 and 3.6% mannitol. The regeneration of protoplasts into viable plants required media optimization, the inclusion of plant growth regulators and the correct culture technique. Microcalli derived from protoplasts were obtained by establishing agarose bead cultures using Y3A medium supplemented with 10μM naphthalene acetic acid, 2μM 2,4-dichlorophenoxyacetic acid, 2μM indole-3-butyric acid, 2μM gibberellic acid and 2μM 2-γ-dimethylallylaminopurine. Small plantlets were regenerated from microcalli by somatic embryogenesis after successive subculturing steps in medium with limiting amounts of growth regulators supplemented with 200mg/l ascorbic acid.

Development of transformation vectors for the production of potentially high oleate transgenic oil palm

The main target of Malaysian Palm Oil Board (MPOB) genetic engineering programme is to produce high oleate transgenic palms. The availability of effective transformation vector is one of the pre-requisites for genetic manipulation of oil palm through recombinant DNA technology. Here, we describe the construction of a series of transformation vectors that have a maize ubiquitin promoter ( UbiPro )-driven bar gene for selection of transformants on herbicide (Basta or Bialaphos), and mesocarp-specific promoter ( MSP 1) for expression of the transgenes [antisense palmitoyl-ACP-thioesterase ( PAT ) and sense β-ketoacyl-ACP-synthase II ( KAS II) and sense D9-stearoyl-ACP-desaturase ( SAD )] potentially responsible for high oleate content in oil palm mesocarp. The transformation vectors constructed in this study are suitable for use in both particle bombardment (biolistic) and Agrobacterium -based transformation protocols.