Production of Specialized Transformation Vectors for the Production of Biodegradable Plastics in Transgenic Arabidopsis and Oil Palm

Polyhydroxyalkanoates (PHAs), such as polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate (PHBV) are bacterial polyesters, which can be used to produce biodegradable products. Since the mass production of PHAs in bacteria via fermentation is expensive, the production of PHAs in plants may be an attractive alternative. The production of PHB in plants required genetic engineering of phbA, phbB and phbC genes of Ralstonia eutropha, whereas, the bktB, phbB, phbC genes of R. eutropha and tdcB gene of Escherichia coli were required for PHBV production. In this study, each of these gene was fused with the transit peptide (Tp) of oil palm acyl-carrier-protein (ACP), and driven by the oil palm leaf-specific promoter (LSP1), for targeting into the plastids of leaf cells. In total, four transformation vectors, pLSP15 (PHB) and pLSP20 (PHBV), pLSP13 (PHB) and pLSP23 (PHBV) were constructed for the transformation of Arabidopsis and oil palm, respectively. Each vector contained the phosphinothricin acetyltransferase gene (Bar) driven by CaMV35S promoter in pLSP15 and pLSP20, and ubiquitin promoter in pLSP13 and pLSP23, as plant selectable marker. Matrix attachment region of tobacco (RB7MAR) was also included, to stabilize the transgene expression and to minimize gene silencing due to positional effects. Restriction enzymes, polymerase chain reaction (PCR) and DNA sequencing were used to verify all the constructed vectors. Arabidopsis transformation produced T1 transgenic Arabidopsis plants with normal phenotypes at a transformation efficiency of 0.2%~1.0%. PCR and Southern analyses were used to confirm the insertion of the transgenes. Nile blue A staining of these T1 plants demonstrated the accumulation of PHB granules in the leaf. The initial screening of Basta-resistant oil palm embryogenic calli transformed with pLSP13 using PCR demonstrated the presence of Bar and PHB genes in transformed oil palm

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

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

Efficient protocol improved the yield and viability of oil palm protoplasts isolated from in vitro leaf and mesocarp

The absence of a cell wall on the protoplast contributes to its versatility. Its flexibility for DNA manipulation and the possibility of rapid cell-based assay is desirable in the plant biotechnology field. This study was carried out to improve protoplast isolation from oil palm in vitro leaf and mesocarp tissues. The factors affecting protoplast isolation efficiency were optimized, including the protocols and enzyme composition involved, focusing on the oil palm in vitro leaf first. Incubation of oil palm leaf sample with an enzyme mixture of cellulase R-10, macerozyme R-10, driselase, and pectolyase Y-23, for 14 h has successfully produced up to 2.5 × 106 protoplasts g-1 fresh weight (FW)-1 with 95% viability. Incubation of oil palm mesocarp tissue with the optimized enzyme mixture for 2 h at static condition has also successfully produced 3.98 × 106 protoplasts g-1 FW-1 with 85% viability. Besides, it was found that increasing the sample's surface area in contact with enzyme solution by slicing the samples into narrow strips and thin layers has improved the penetration of enzymes into the tissues and enhanced the isolation efficiency. In addition, a plasmolysis step before enzymatic treatment has also improved the protoplast viability by minimizing the damage incurred during isolation. The successful isolation of protoplast from oil palm leaf and mesocarp has enabled the study of gene function and the characterization of endogenous tissue-specific promoter being carried out in vivo

Development of a protoplast based transformation system for genetic engineering of oil palm

The major aim of the thesis was to develop the prerequisites for efficient genetic engineering of oil palm by DNA microinjection with the long-term objective to generate transgenic oil palm producing recombinant proteins, PIPP (a chimeric antibody against human chorionic gonadotropin; hCG), D12 (a human antibody against dental carries) and HSA (human serum albumin). The products will be synthesized in the leaf, mesocarp and kernel tissues of oil palm with the respects of plants must be stable and free from selectable marker. To achieve this, the constructs of PIPP, D12 and HSA genes, which were driven either by the promoter of LSP, MSP or KSP were successful constructed and their functionality was demonstrated in tobacco plants. To implement the oil palm protoplasts as starting material for the development of stable transgenic oil palms via DNA microinjection, the regeneration of true plants from protoplasts is a mandatory. Therefore, an improved protocol for the efficient isolation of high-quality protoplasts from oil palm suspension cultures was established. Subsequently, for the first time true oil palms were successfully regenerated from oil palm protoplasts by using optimal parameters. Nearly 14-17 months after protoplasts were isolated; true plants were generated using agarose bead culture. Following the success in regeneration of plants from protoplasts, the objective of this project became clearly to be achieved in the future when the protoplasts were used for PEG-mediated transient gene expression, and further used in the stable gene expression via DNA microinjection. The efficient and reliable protocol for PEG mediated transformation of oil palm protoplasts was developed by determing and validating the optimal parameters like heat shock treatment, the amount of DNA, PEG and magnesium chloride concentrations, and the procedure to transfect the protoplasts. As the main objective of this study, the transgenic microcalli of oil palm were successful generated from protoplasts transformed by DNA microinjection within 6 months. More conclusive results will be obtained when small plantlets are produced and analyzed

Development of a protoplast based transformation system for genetic engineering of oil palm

The major aim of the thesis was to develop the prerequisites for efficient genetic engineering of oil palm by DNA microinjection with the long-term objective to generate transgenic oil palm producing recombinant proteins, PIPP (a chimeric antibody against human chorionic gonadotropin; hCG), D12 (a human antibody against dental carries) and HSA (human serum albumin). The products will be synthesized in the leaf, mesocarp and kernel tissues of oil palm with the respects of plants must be stable and free from selectable marker. To achieve this, the constructs of PIPP, D12 and HSA genes, which were driven either by the promoter of LSP, MSP or KSP were successful constructed and their functionality was demonstrated in tobacco plants. To implement the oil palm protoplasts as starting material for the development of stable transgenic oil palms via DNA microinjection, the regeneration of true plants from protoplasts is a mandatory. Therefore, an improved protocol for the efficient isolation of high-quality protoplasts from oil palm suspension cultures was established. Subsequently, for the first time true oil palms were successfully regenerated from oil palm protoplasts by using optimal parameters. Nearly 14-17 months after protoplasts were isolated; true plants were generated using agarose bead culture. Following the success in regeneration of plants from protoplasts, the objective of this project became clearly to be achieved in the future when the protoplasts were used for PEG-mediated transient gene expression, and further used in the stable gene expression via DNA microinjection. The efficient and reliable protocol for PEG mediated transformation of oil palm protoplasts was developed by determing and validating the optimal parameters like heat shock treatment, the amount of DNA, PEG and magnesium chloride concentrations, and the procedure to transfect the protoplasts. As the main objective of this study, the transgenic microcalli of oil palm were successful generated from protoplasts transformed by DNA microinjection within 6 months. More conclusive results will be obtained when small plantlets are produced and analyzed