Transgenic Oil Research Articles

Transcription factor EgGRP2A regulates EgFATA expression and promotes oleic acid accumulation in oil palm (Elaeis guineensis)

The oil palm (Elaeis guineensis) is emerging as the world's most important and prolific oilseed crop, celebrated for its impressive oil yield. However, the molecular intricacies that govern lipid metabolism and fatty acid accumulation in oil palm fruits remain relatively underexplored. This study reveals a significant correlation between the expression of EgGRP2A, a transcription factor, and the expression of EgFATA in the oil palm. Yeast one-hybrid analysis and electrophoretic mobility shift assays (EMSA) reveal and confirm the binding interactions between EgGRP2A and the promoter region of EgFATA. Subsequent experiments in oil palm protoplasts show that transient overexpression of EgGRP2A leads to a marked upregulation of EgFATA expression. Conversely, downregulation of EgGRP2A in transgenic oil palm embryoids leads to a significant reduction in EgFATA expression. Metabolite profiling in the transgenic embryoids reveals a significant reduction in unsaturated fatty acids, particularly oleic acid. These findings promise profound insights into the regulatory orchestration of EgFATA and the synthesis of fatty acids, particularly oleic acid, in the oil palm. Furthermore, the results lay the foundation for future breeding and genetic improvement efforts aimed at increasing oleic acid content in oil palm varieties.

Oils from Transgenic Flax Lines as Potential Chemopreventive Agents in Colorectal Cancer.

Colorectal cancer is a major global health concern, and the need for effective chemopreventive agents is paramount. This study aimed to evaluate the potential of oils from transgenically modified flax for the prevention of colorectal cancer, in relation to the oil concertation. Flaxseed oils were obtained from traditional (Nike) and genetically modified flax lines (M and B). Cell viability assays were performed on various cancer cell lines, including colon adenocarcinoma cells. Flaxseed oil B exhibited the strongest anti-proliferative properties compared to the reference drugs and other oils. Additionally, M and B oils showed enhanced accumulation of Rhodamine 123 and increased apoptosis in colorectal cancer cells. M oil exhibited the highest levels of p53 protein. Notably, the tested transgenic oils did not induce metastasis and displayed stronger inhibition of COX-1 compared to COX-2. These data indicate the utility of flaxseed oils, especially from the M line, as adjuvants in colorectal cancer treatment, targeting the colon specifically.

Defining the physical properties of blends of acetyl‐triacylglycerols derived from transgenic oil seeds

AbstractAcetyl‐TAG are unusually structured triacylglycerols (TAG) that possess an acetate group at their sn‐3 position. Acetyl‐TAG have different physical properties compared to regular TAG, including lower viscosity and improved cold temperature properties, making acetyl‐TAG useful for different applications, including as a diesel replacement. These unusual TAG molecules can be synthesized in the seeds of transgenic plants through the expression of diacylglycerol acetyltransferase (DAcT) enzymes isolated from different Euonymus species. In this study, the kinematic viscosity as well as the crystallization and melting behavior of blends of acetyl‐TAG and regular TAG were examined to define goals for acetyl‐TAG synthesis in transgenic plants. Even small amounts of regular TAG when blended with acetyl‐TAG had a disproportionate effect on the viscosity of mixture. This effect of regular TAG in increasing kinematic viscosity was more pronounced at lower temperatures. Under slow cooling conditions, the two different TAGs and their blends possessed two main crystallization events with different degree of separation of the thermal transition, and the lower crystallization temperature decreased with increasing amounts of acetyl‐TAG in the blend. At higher cooling rates, one broad and tailed crystallization peak was observed. Heating thermograms indicate similar polymorphic behavior of the blends and a general peak shift to lower transition range with increasing acetyl‐TAG compared to the two pure lipids. This information about the viscosity and thermal properties of blends of TAG and acetyl‐TAG will provide useful targets for engineering higher levels of acetyl‐TAG in transgenic seeds.

A review on the identification of transgenic oilseeds and oils.

Transgenic technology can increase the quantity and quality of vegetable oils worldwide. However, people are skeptical about the safety of transgenic oil-bearing crops and the oils they produce. In order to protect consumers' rights and avoid transgenic oils being adulterated or labeled as nontransgenic oils, the transgenic detection technology of oilseeds and oils needs careful consideration. This paper first summarized the current research status of transgenic technologies implemented at oil-bearing crops. Then, an inspection process was proposed to detect a large number of samples to be the subject rapidly, and various inspection strategies for transgenic oilseeds and oils were summarized according to the process sequence. The detection indicators included oil content, fatty acid, triglyceride, tocopherol, and nucleic acid. The detection technologies involved chromatography, spectroscopy, nuclear magnetic resonance, and polymerase chain reaction. It is hoped that this article can provide crucial technical reference and support for staff engaging in the supervision of transgenic food and for researchers developing fast and efficient monitoring methods in the future.

An Efficient Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-Associated Protein 9 Mutagenesis System for Oil Palm (Elaeis guineensis).

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful tool for the precise editing of plant genomes for crop improvement. Rapid in vitro methods for the determination of guide RNA (gRNA) cleavage efficiency and an efficient DNA delivery system is essential for gene editing. However, we lack an efficient gene-editing system for palm species. In this study, we described the development of a transient oil palm protoplast assay to rapidly evaluate the cleavage efficiency of CRISPR/Cas9 mutagenesis and the generation of stable transformed oil palms using biolistic particle bombardment in immature embryos. Using the phytoene desaturase (EgPDS) gene, we found cleavage frequency of up to 25.49% in electro-transfected protoplast, which enables the production of transgenic oil palm shoots exhibiting chimeric albino phenotypes as a result of DNA insertions, deletions (InDels), and nucleotide substitutions, with a mutation efficiency of 62.5–83.33%. We further validated the mutagenesis efficiency and specificity of the CRISPR/Cas9 system in oil palm by targeting the brassinosteroid-insensitive 1 (EgBRI1) gene, which resulted in nucleotide substitutions in EgBRI1 with premature necrosis phenotype in oil palm transgenic shoots and stunted phenotype resulting from DNA InDels. Taken together, our results showed that effective and efficient editing of genes using the CRISPR/Cas9 system can be achieved in oil palm by optimizing the selection of efficient gRNA and DNA delivery methods. This newly designed strategy will enable new routes for the genetic improvement in oil palm and related species.

Transgenic Camelia sativa oil: an effective source of both EPA and DHA in the human diet

Transgenic Camelia sativa oil: an effective source of both EPA and DHA in the human diet

Physiological responses and P5CS gene expression of transgenic oil palm plantlet induced by drought stress

Drought is one of the limiting factors in crop cultivation, such as in oil palm (Elaeis guineensis Jacq.). The transgenic approaches are expected to increase plant tolerance to drought stress and minimize low productivity when drought occurs. Proline is an osmoprotectant compound in plants which its biosynthesis involved the P5CS gene. The objective of this study was to evaluate the tolerance level of P5CS-transgenic oil palm to drought stress induced by polyethylene glycol 6000 (PEG-6000). In this present study, the transgenic and non-transgenic oil palms were treated by 0, 2, and 4% PEG-6000 under in vitro conditions. The experiment was arranged as a factorial completely randomized design with three replications. The drought level score, total chlorophyll content, carotenoids, and proline content, as well as P5CS gene expression in leaf tissues were observed at 7 and 14 days after stress treatments. The result showed that transgenic plantlets had a lower drought level score than those of non-transgenic lines. A concentration of 4% PEG-6000 treatment reduced the total chlorophyll and carotenoids contents than that of 2% concentration in non-transgenic plantlets at 7 and 14 day after treatments (DAT). In addition, proline content and P5CS gene expression level in transgenic had been significantly increased during stress treatment. Based on these results, it can be concluded that the P5CS transgene increased the drought stress tolerance of oil palm.

Molecular Pharming of the Recombinant Protein hEGF-hEGF Concatenated with Oleosin Using Transgenic Arabidopsis.

We set out to assess the NIH/3T3 cell proliferation activity of Arabidopsis oil body-expressed recombinant oleosin–hEGF–hEGF protein. Normally, human epidermal growth factor (hEGF) is purified through complex process, however, oleosin fusion technology provides an inexpensive and scalable platform for its purification. Under a phaseolin promoter, we concatenated oleosin gene to double hEGF (hEGF–hEGF) with plant-preferred codons in the expression vectors and the construct was transformed into Arabidopsis thaliana (Arabidopsis). The transgenic Arabidopsis was validated by RT–PCR and the content of recombinant protein oleosin–hEGF–hEGF was quantified by western blot. Subsequently, the proliferation assay and transdermal absorption were determined by MTT method and immunohistochemical staining, respectively. First, the expression level of hEGF was recorded to be 14.83-ng/μL oil body and due to smaller size transgenic oil bodies expressing the recombinant oleosin–hEGF–hEGF, they were more skin permeable than those of control. Second, via the staining intensity of transgenic oil bodies was greater than EGF at all time points via immunohistochemical staining in transdermal absorption process. Lastly, activity assays of oil bodies expressed oleosin–hEGF–hEGF indicated that they stimulated the NIH/3T3 cell proliferation activity. Our results revealed oil-body-expressed oleosin–hEGF–hEGF was potential new material having implications in the field of medicine.

A MADS-box gene, EgMADS21, negatively regulates EgDGAT2 expression and decreases polyunsaturated fatty acid accumulation in oil palm (Elaeis guineensis Jacq.).

EgMADS21 regulates PUFA accumulation in oil palm. Oil palm (Elaeis guineensis Jacq.) is the most productive world oil crop, accounting for 36% of world plant oil production. However, the molecular mechanism of the transcriptional regulation of fatty acid accumulation and lipid synthesis in the mesocarp of oil palm by up- or downregulating the expression of genes involved in related pathways remains largely unknown. Here, an oil palm MADS-box gene, EgMADS21, was screened in a yeast one-hybrid assay using the EgDGAT2 promoter sequence as bait. EgMADS21 is preferentially expressed in early mesocarp developmental stages in oil palm fruit and presents a negative correlation with EgDGAT2 expression. The direct binding of EgMADS21 to the EgDGAT2 promoter was confirmed by electrophoretic mobility shift assay. Subsequently, transient expression of EgMADS21 in oil palm protoplasts revealed that EgMADS21 not only binds to the EgDGAT2 promoter but also negatively regulates the expression of EgDGAT2. Furthermore, EgMADS21 was stably overexpressed in transgenic oil palm embryoids by Agrobacterium-mediated transformation. In three independent transgenic lines, EgDGAT2 expression was significantly suppressed by the expression of EgMADS21. The content of linoleic acid (C18:2) in the three transgenic embryoids was significantly decreased, while that of oleic acid (C18:1) was significantly increased. Combined with the substrate preference of EgDGAT2 identified in previous research, the results demonstrate the molecular mechanism by which EgMADS21 regulates EgDGAT2 expression and ultimately affects fatty acid accumulation in the mesocarp of oil palm.

Advance of Multiplex PCR in Rapid Detecting Transgenic Soybean Oil

Transgenic food safety is a high-profile public health issue in worldwide, especially transgenic soybean (Glycine max L.) oil. To rapidly and effectively detect transgenic components of soybean oil, in the present study, we isolated DNA from transgenic soybean oil by modified method, and employed the multiplex PCR method to identify targeted genes, including CaMV35S promoter, Nos terminator, NPTII, CP4-EPSPS and endogenous gene Lectin. The research aims to build a method which is accurate, rapid and reliable for detection of genetically modified soybeans oil. The targeted gene including DNA was successfully established by the improved method, and then amplified by PCR. Five genes are simultaneously specifically detected. Commercial soybean, genetically modified soy bean and oil were detected with the Multiplex PCR. The improved method of DNA extraction was rapid and accurate to extract high quality total DNA which was amplified by PCR. The method could eliminate the PCR inhibitor. A way of detecting the genetically modified soybean and Oil was set up in this study.

Biolistic-mediated oil palm transformation with alfalfa glucanase (AGLU1) and rice chitinase (RCH10) genes for increasing oil palm resistance towards Ganoderma boninense

Basal stem rot (BSR) caused by Ganoderma boninense, a white-rot fungus is the most devastating disease in oil palm. In addition to the available methods that have been used to control this disease, genetic engineering was explored to produce fungal resistant transgenic oil palm. In this study, oil palm embryogenic calli were co-bombarded with a construct carrying the AGLU1 and RCH10 genes and a construct carrying the Basta® resistance gene (bar). After selection with the herbicide Basta®, 25 plants from seven events were regenerated, transferred onto soil and grown in the fully contained biosafety screen house. Polymerase chain reaction (PCR) analysis for the plants demonstrated that only eight plants were positive for the presence of three transgenes, the bar, AGLU1, and RCH10 genes. The quantitative reverse transcription PCR (RT-qPCR) demonstrated the expression of both AGLU1 and RCH10 genes in five out of eight plants tested. The infection of these plants with the G. boninense fungus resulted in only two plants exhibited resistance to the fungus. The results from this study demonstrated that fungal resistant oil palm could be produced via biolistic using alfalfa β-1,3-glucanase (AGLU1) and rice chitinase (RCH10) genes.

Evaluation of transient DsRED gene expression in oil palm embryogenic calli

Genetic modification of oil palm is being carried out to explore new avenues for improving and expanding the potential of oil palm. Multiple bottlenecks in delivering the DNA into oil palm cells and generating the transgenic oil palm required us to look into an alternative marker which could help us assess the efficiency of the transformation system, moreover at the early stage considering that oil palm is a slow growing plant. In this study, we have constructed seven transformation vectors carrying a gene encoding DsRED, each driven by a different promoter, i.e. plant constitutive promoter, oil palm constitutive promoter or oil palm tissue-specific promoter. The vectors were transformed into oil palm embryogenic calli via microprojectile bombardment and the red fluorescence signals were observed after 48 h. Based on the observation, the construct driven by the CaMV35S promoter produced the highest number of red fluorescent spots. The oil palm calli bombarded with pUbi-DsRED plasmid suffered the most number of red fluorescent spots losses from day three until day 42, as compared to other constructs. The red fluorescent signals of DsRED produced across all transformed samples appeared to be bright and very distinctive as compared to the background.

Rapid and accurate detection of genetically manipulated soybean using polymerase chain reaction

Background: It is controversy over the GM products consumption around the world. However, the limitations of food supplies and population growth have forced the posterity to use transgenic products. Accurate strategies for consumption and use of proper diagnostic methods are crucial in this regard. Materials and methods: The soybean commercial samples were purchased as a control from supermarkets located in Tehran. Considerable attention was paid to collecting samples from different brands. In this investigation, in order to identify the soybean oil in products, randomly, 25 raw oil and soybean oil samples were collected and numbered. An equivalent of 50 g of each sample was prepared for testing. In order to determine the transgenic soya samples, the specific primers for the 35S promoter region of mosaic virus CaMV35S and nephaline synthase (NOS) transcription terminator were designed from Agrobacterium. The sequence of the CaMV35S promoter and the NOS terminator were obtained according to that of Lipp and Brodmann investigation. The PCR product size for 35S and NOS primers was 195 and 180 bp, respectively. Results: By PCR technique the specific Soybean (164bp), GMONOS180 (180bp) and GMO35S (195bp) genes were amplified. Among the 25 tested oils, 9 (36%) were GM, being a high percent and of these nine transgenic cases, two had CaV-35s, and also were nos-positive, indicating of their transfusion certainty (GM). This high percentage of transgenic oils is possibly due to the lack of awareness of the producers. Conclusion: The microbiological and chemical methods alone cannot control the quality of food products, and detect the presence of transgenes. Therefore, the use of PCR technique is important in controlling the quality of this product. In cases where manufacturers and importers of products prepare and distribute transgenic products without labeling, they can be easily prevented from misuse and thus PCR as complementary and helpful to determine the composition and quality of the product.

Embryogenic callus induction and fatty acid composition analysis of oil palm (Elaeis guineensis cv. Tenera)

Embryogenic callus (EC) induction and plantlet regeneration of oil palm (Elaeis guineensis) are essential platforms for the propagation of uniform hybrid cultivars in large-scale cultivation. In this paper, an embryogenic callus was successfully induced from leaf tissue from the ‘Tenera’ varieties of oil palm and regenerated into plantlets via secondary embryogenesis. The total lipid and separated triacylglycerol (TAG) contents of the EC were determined using GC–MS. The results showed that the EC had a total lipid content of approximately 9.46%, while TAG accounted for 32% of the total lipid content. Considering that the TAG content was more than 95% of the total fatty acids (FAs) in the mesocarp of ripe oil palm fruit, the FA profile of the TAG content in the EC was compared with the total lipid content in the mesocarp of oil palm. The results indicated that the FA profile of the TAG content in the EC was similar to that in the mesocarp, even though there were still significant differences in the content of individual FAs. The similar FA composition in the EC and mesocarp of oil palm will be a valuable marker for predicting FA profiles in the mesocarp of the mature transgenic oil palm fruit obtained from transformation and embryogenesis.

Green fluorescent protein as a visual selection marker for oil palm transformation

The aim of the study was to regenerate transgenic oil palm expressing the gfp gene throughout the whole plant by transforming oil palm cultures with organelle-targeted and non-organelle targeted gfp genes. In this study, oil palm embryogenic calli and immature embryos were separately bombarded with different constructs carrying either organelle-targeted (mitochondria, plastid or endoplasmic reticulum) or non-organelle targeted gfp gene in combination with the herbicide Basta resistance (bar) gene for regenerating transgenic oil palm expressing gfp. Transformed embryogenic calli were successfully regenerated on media containing Basta but failed to express the gfp gene in the regenerated plants. On the other hand, the bombarded immature embryos were either directly germinated or regenerated into whole plantlets via callogenesis on medium containing Basta. The regenerated plantlets demonstrated gfp expression only as patches on some parts of the plants such as roots and leaves and not in the whole plant, contrary to what was expected. Even though the plantlets failed to show gfp gene expression in the whole plant, molecular analyses revealed the presence of bar and gfp transgenes in the transgenic plants. Based on results obtained from this study, it could be concluded that the failure was due to the inability of gfp gene to express in the regenerated transgenic plants. In order to overcome this failure, further interventions are proposed which include the use of stronger promoters, use of different gfp versions and finally to use alternative transformation methods.

BIOTECHNOLOGY FOR DIVERSIFICATION AND IMPROVED RESILIENCE OF THE OIL PALM

Oil palm is one of the most important crops in Malaysia and has brought about significant economic and social development to the country. Nevertheless, there are major drawbacks and challenges faced by the industry such as shrinking availability of arable land, labour issues, and pest and disease. This has forced the industry to be more proactive by increasing yield and venturing into synthesis of higher value products. Besides conventional breeding which has made a lot of contributions, biotechnology has been identified as one of the tools to overcome the mentioned challenges. Oil palm biotechnology, especially genetic modification, began more than three decades ago leveraging multidisciplinary research including biochemistry of lipid biosynthesis, isolation of useful genes and promoters and finally genetic transformation. The main target of MPOB’s oil palm genetic modification effort is to produce palms with higher oleic acid content. Other targets are synthesising palmitoleic acid and ricinoleic acid, increasing stearic acid and lycopene (carotenoid) content and production of biodegradable plastics. Biotechnological research also targets towards better understanding the pathogen biology and disease epidemiology for implementing disease management programmes. In the area of disease management, especially for Ganoderma species, study on plant pathogenesis and plant-microbe interaction is also being carried out. Significant achievements have resulted from biochemical studies, isolation and characterisation of important oil palm genes and promoters. Furthermore, transformation of constructs with various targetted products into oil palm embryogenic calli was successfully carried out while the regeneration of transgenic oil palm harbouring the useful genes is in progress. Towards developing oil palm with pest and disease tolerance, efforts have been initiated to understand the oil palm defence mechanisms. Several differentially expressed defence-related genes governing the interaction of the plant pathogen and oil palm have been identified. Besides understanding the oil palm system, research into the pathogen Ganoderma including identification of pathogen strains, developing various markers and data mining of fungal genome sequences have been conducted. Keywords: Genetic modification, promoters, lipid biosynthesis,carotenoid, Ganoderma.

Terahertz spectroscopy and chemometrics classification of transgenic corn oil from corn edible oil

ABSTRACTTerahertz spectroscopy coupled with chemometrics is illustrated to be a useful tool to identify the transgenic corn oil. Terahertz and physicochemical data are gather about transgenic and non‐transgenic corn oils. The two identification models of partial least squares discriminate analyses (PLS‐DA) are established and compared by using terahertz spectroscopy data and physicochemical data respectively. The result shows that PLS‐DA model with physicochemical data can not accurately distinguish between transgenic and non‐transgenic corn oils. Quite the contrary, the PLS‐DA model with terahertz spectroscopy data is better in the validation set; this model accurately identified transgenic corn oil with accuracy of 98.7%. The proposed method has advantages of fast, simple, nondestructive, and easy to operate. The results indicated that Terahertz spectroscopy coupled with chemometrics as a powerful tool to identify transgenic oil. © 2017 Wiley Periodicals, Inc. Microwave Opt Technol Lett 59:654–658, 2017

基于太赫兹时域光谱技术与PCA-SVM的转基因大豆油鉴别研究

基于太赫兹时域光谱技术与PCA-SVM的转基因大豆油鉴别研究

MCR-ALS and NIRS applied on the evaluation of the Lepidium meyenii antioxidant activity

The Peruvian Maca (Lepidium Meyenii Walp) is a food which is widely consumed around the Peruvian Andes and has been used in Brazil thanks to medicinal properties attributed by popular culture. Due to lack of information on this cultivar, it was proposed a study to evaluate the effect of adding Peruvian Maca in transgenic soybean oil. For this purpose, the spectra of soybean oil samples with and without addition of Maca after heating at different temperatures were measured at Visible and Near Infrared regions. The results were evaluated by chemometric methods of Principal Component Analysis (PCA) and Multivariate Curve Resolution with Alternating Least Squares (MCR-ALS). The results suggest that Maca inhibits the appearance of oxidation products, which is evidenced when comparing the results obtained for the heated oil with and without addition of Maca.

Future availability of raw materials for salmon feeds and supply chain implications: The case of Scottish farmed salmon

The current range of Scottish salmon feeds is adapted to a differentiated supply of salmon products, including differing omega-3 content, differing content of marine raw materials, etc. The progressive replacement of marine feed ingredients by plant proteins and oils is reducing the content of omega-3 long-chain polyunsaturated fatty acids (LC-PUFA). However the benefits are a more secure and less volatile raw material supply, together with environmental feed contaminants at low or undetectable levels in the resulting salmon product. There is widespread adoption of standards and certification schemes by Scottish salmon farmers and feed suppliers in order to demonstrate environmental sustainability. This has focused in particular on use of certified ingredients from sustainable supply sources (‘responsible sourcing’). Future volume estimates of Scottish salmon production, hence feed requirements, are insufficient to threaten raw material supply compared with global markets, although it is argued this is likely to involve greater use of locally grown plant proteins and an increased proportion of fishmeal manufactured from by-product trimmings (derived from processing fish for human consumption). However, UK retail chains will remain reluctant to allow salmon suppliers to utilise land animal by-products due to negative consumer perceptions, with resulting implications for formulation cost and flexibility. Given its world-wide scarcity, the main strategic concern relates to future availability of sufficient omega-3 LC-PUFA, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in order to maintain the healthy image of Scottish salmon. To maintain its longer-term reputation and product benefits, the Scottish industry may need to consider adopting a more flexible attitude to using new alternatives to fish oil (e.g. EPA and DHA derived from transgenic oil seed crops, when commercially available). It is concluded that Scottish salmon farming is a successful example of sustainable feed development and the industry can be confident that the changing raw material base will support continuing production of high quality, healthy farmed salmon, but the long-term security of supply of omega-3 LC-PUFA remains an issue.