Reporter gene activity under the regulation of the oil palm metallothionein-like gene, MT3-A promoter was assessed in prokaryotes. Vector constructs containing MT3-A promoter with (W1MT3-A) and without (W2MT3-A) five prime untranslated region (5'-UTR) fused to ß-glucuronidase (GUS) gene in pCAMBIA 1304 vector were produced. 5'-rapid amplification of cDNA ends (RACE) using mRNA isolated from Escherichia coli and Agrobacterium tumefaciens harboring W1MT3-A confirmed that fusion transcripts of MT3-A 5'-UTR-GUS were successfully produced in both bacteria. Competitive PCR and GUS fluorometric assay showed changes in the level of GUS gene transcripts and enzyme activity in response to increasing concentrations of Cu²+ and Zn²+. The application of Cu²+ increased GUS activity and GUS mRNA level in both bacteria. In E. coli, a high level of GUS activity driven by W1MT3-A and W2MT3-A was observed in treatment with 25 μM Cu²+ resulting in an increase in the GUS mRNA level to 7.2 and 7.5 x 10⁻⁴ pmol/μl respectively, compared to the control (5.1 x 10⁻⁴ pmol/μl). The lowest GUS activity and GUS mRNA level were obtained for W1MT3-A and W2MT3-A in the presence of 100 μM Cu²+ in both bacteria compared to the control (without Cu²+). The application of different Zn²+ concentrations resulted in a strong decrease in the GUS activity and GUS mRNA level in E. coli and A. tumefaciens. These findings showed that the oil palm MT3-A promoter is functional in prokaryotes and produced detectable GUS transcripts and enzyme activities. This promoter may potentially be used in prokaryotic systems which require metal inducible gene expression.
Physical and biological parameters affecting DNA delivery into oil palm embryogenic calli using the biolistic device are optimized. Five different promoters are also evaluated to identify the most suitable promoter for use in oil palm transformation. Finally, the effectiveness of kanamycin, geneticin (G418), neomycin, hygromycin, and herbicide Basta as selection agents to inhibit growth of oil palm embryogenic calli is evaluated. Combination of optimized parameters, best promoter and selection agent is later used to transform oil palm embryogenic calli for producing transgenic oil palm plants. Bombarded embryogenic calli are exposed to 50 mg/l of Basta after 3 weeks. Basta-resistant embryogenic calli started to emerge five to six months in medium containing Basta. The Basta-resistant embryogenic calli are proliferated until they reach a specific size, and the Basta-resistant calli are later individually isolated and regenerated to produce complete plantlets. The complete regenerated plantlets are evaluated for the presence of transgenes by PCR, Southern and thin layer chromatography analyses.
The use of acetosyringone in Agrobacterium-mediated gene transfer into plant hosts has been favored for the past few decades. The influence of other phenolic compounds and their effectiveness in Agrobacterium-mediated plant transformation systems has been neglected. In this study, the efficacy of four phenolic compounds on Agrobacterium-mediated transformation of the unicellular green alga Nannochloropsis sp. (Strain UMT-M3) was assessed by using β-glucuronidase (GUS) assay. We found that cinnamic acid, vanillin and coumarin produced higher percentages of GUS positive cells as compared to acetosyringone. These results also show that the presence of methoxy group in the phenolic compounds may not be necessary for Agrobacterium vir gene induction and receptor binding as suggested by previous studies. These findings provide possible alternative Agrobacterium vir gene inducers that are more potent as compared to the commonly used acetosyringone in achieving high efficiency of Agrobacterium-mediated transformation in microalgae and possibly for other plants.
Modification of lipid composition in the mesocarp tissue of oil palm involves genetic manipulation of multiple genes. More than one mesocarp-preferential promoter is necessary for the expression of individual transgenes in the same plant to obviate transcriptional gene silencing. This study aimed to identify genes that are preferentially expressed in the mesocarp tissue and characterize selected candidate mesocarp-preferential promoters. Ten transcripts that were preferentially expressed in the mesocarp tissue were identified from the analysis of 82 transcriptome datasets of 12 different oil palm tissues. The expression of two candidate genes, MSP-C1 and MSP-C6, was verified to be preferentially expressed in the mesocarp tissues and shown to have a low expression level in non-mesocarp tissues by reverse transcription quantitative real-time PCR (RT-qPCR). MSP-C6 promoter fragments of different lengths were transformed into tomato plants for further characterization. Both unripe and ripe fruits of transgenic tomato plants transformed with a construct harboring the MSP-C6-F1 (2014 bp) promoter were shown to have high beta-glucuronidase (GUS) activities. The findings of this study suggest the potential applications of the MSP-C6 promoter as a molecular tool for genetic engineering of novel traits in fruit crops.
The successful establishment of an Agrobacterium-mediated transformation method and optimisation of six critical parameters known to influence the efficacy of Agrobacterium T-DNA transfer in the unicellular microalga Chlorella vulgaris (UMT-M1) are reported. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1304 containing the gfp:gusA fusion reporter and a hygromycin phosphotransferase (hpt) selectable marker driven by the CaMV35S promoter were used for transformation. Transformation frequency was assessed by monitoring transient β-glucuronidase (GUS) expression 2 days post-infection. It was found that co-cultivation temperature at 24°C, co-cultivation medium at pH 5.5, 3 days of co-cultivation, 150 μM acetosyringone, Agrobacterium density of 1.0 units (OD(600)) and 2 days of pre-culture were optimum variables which produced the highest number of GUS-positive cells (8.8-20.1%) when each of these parameters was optimised individually. Transformation conducted with the combination of all optimal parameters above produced 25.0% of GUS-positive cells, which was almost a threefold increase from 8.9% obtained from un-optimised parameters. Evidence of transformation was further confirmed in 30% of 30 randomly-selected hygromycin B (20 mg L(-1)) resistant colonies by polymerase chain reaction (PCR) using gfp:gusA and hpt-specific primers. The developed transformation method is expected to facilitate the genetic improvement of this commercially-important microalga.
KEY MESSAGE: The oil palm EgPAL1 gene promoter and its regulatory region were functional as a promoter in the heterologous system of Arabidopsis according to the cis-acting elements present in that region. The promoter was developmentally regulated, vascular tissue specific and responsive to water stress agents. Phenylalanine ammonia lyase (PAL, EC 4.3.1.24) is the key enzyme of the phenylpropanoid pathway which plays important roles in plant development and adaptation. To date, there is no report on the study of PAL from oil palm (Elaeis guineensis), an economically important oil crop. In this study, the 5' regulatory sequence of a highly divergent oil palm PAL gene (EgPAL1) was isolated and fused with GUS in Arabidopsis to create two transgenic plants carrying the minimal promoter with (2302 bp) and without its regulatory elements (139 bp). The regulatory sequence contained cis-acting elements known to be important for plant development and stress response including the AC-II element for lignin biosynthesis and several stress responsive elements. The promoter and its regulatory region were fully functional in Arabidopsis. Its activities were characterised by two common fundamental features of PAL which are responsive to plant internal developmental programme and external factors. The promoter was developmentally regulated in certain organs; highly active in young organs but less active or inactive in mature organs. The presence of the AC elements and global activity of the EgPAL1 promoter in all organs resembled the property of lignin-related genes. The existence of the MBS element and enhancement of the promoter activity by PEG reflected the behaviour of drought-responsive genes. Our findings provide a platform for evaluating oil palm gene promoters in the heterologous system of Arabidopsis and give insights into the activities of EgPAL1 promoter in oil palm.
The stearoyl-acyl-carrier-protein (ACP) desaturase is a plastid-localized enzyme that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays an important role in the determination of the properties of the majority of cellular glycerolipids. Functional characterization of the fatty acid desaturase genes and their specific promoters is a prerequisite for altering the composition of unsaturated fatty acids of palm oil by genetic engineering. In this paper, the specificity and strength of the oil palm stearoyl-ACP desaturase gene promoter (Des) was evaluated in transgenic tomato plants. Transcriptional fusions between 5' deletions of the Des promoter (Des1-4) and the β-glucuronidase (GUS) reporter gene were generated and their expression analyzed in different tissues of stably transformed tomato plants. Histochemical analysis of the Des promoter deletion series revealed that GUS gene expression was confined to the tomato fruits. No expression was detected in vegetative tissues of the transgenic plants. The highest levels of GUS activity was observed in different tissues of ripe red fruits (vascular tissue, septa, endocarp, mesocarp and columella) and in seeds, which harbored the promoter region located between -590 and +10. A comparison of the promoter-deletion constructs showed that the Des4 promoter deletion (314bp) produced a markedly low level of GUS expression in fruits and seeds. Fluorometric analysis of the GUS activity revealed a 4-fold increase in the activity of the full-length Des promoter compared to the CaMV35S promoter. RNA-hybridization analyses provided additional evidence of increased GUS expression in fruits driven by a Des fragment. Taken together, these results demonstrate the potential of the Des promoter as a tool for the genetic engineering of oil palms and other species, including dicots, in improving the quality and nutritional value of the fruits.
Morinda citrifolia, is a valuable medicinal plant with a wide range of therapeutic properties and extensive transformation study on this plant has yet been known. Present study was conducted to establish a simple and reliable transformation protocol for M. citrifolia utilising Agrobacterium tumefaciens via direct seed exposure. In this study, the seeds were processed by tips clipping and dried and subsequently incubated in inoculation medium. Four different parameters during the incubation such as incubation period, bacterial density, temperature and binary vectors harbouring beta-glucuronidase (GUS) gene (pBI121 and pGSA1131), were tested to examine its effect on transformation efficiency. The leaves from the treated and germinated seedlings were analysed via Polymerase Chain Reaction (PCR), histochemical assay of the GUS gene and reverse transcription-PCR (RT-PCR). Results of the study showed that Agrobacterium strain LBA4404 with optical density of 1.0 and 2 h incubation period were optimum for M. citrifolia transformation. It was found that various co-cultivation temperatures tested and type of vector used did not affect the transformation efficiency. The highest transformation efficiency for M. citrifolia direct seed transformation harbouring pBI121 and pGSA1131 was determined to be 96.8% with 2 h co-cultivation treatment and 80.4% when using bacterial density of 1.0, respectively. The transformation method can be applied for future characterization study of M. citrifolia.