Transgenic oil palm: production and projection

The amenability of oil palm (Elaeis guineensis Jacq.) tissues to transgene transfer was first demonstrated more than 10 years ago. Since then several techniques, both direct and vectormediated, were developed to introduce useful genes into different target tissues of oil palm.

Plant Cell Reports, 2014

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.

Methods in Molecular Biology, 2012

Transgenic oil palm (Elaeis guineensis Jacq.) plantlets were regenerated after using Agrobacterium tumefaciens-mediated transformation of embryogenic calli derived from young leaves of oil palm. The calli were transformed with a Agrobacterium strain, LBA4404, harbouring the plasmid pUBA, which carries a selectable marker gene (bar) for resistance to the glufosinate-ammonium and is driven by a maize ubiquitin promoter. Modifications of the transformation method, treatment of the target tissues using acetosyringone, exposure to a plasmolysis medium and physical injury via biolistics were applied. The main reasons for such modifications were to activate the bacterial virulence system and to subsequently increase the transformation efficiency. Transgenic oil palm was selected and regenerated on a medium containing glufosinate-ammonium.

Electronic Journal of Biotechnology, 2008

2007

Green fluorescent protein (GFP) can be easily visualized under UV/blue light without any additional substrate or co-factor. Its assay is also non-destructive. Therefore, it has been widely used to monitor transgene expression and protein localization in variety of cells and organisms. To evaluate the effectiveness and practicality of GFP as a reporter and selectable marker for oil palm transformation, transient expression of GFP genes in oil palm embryogenic calli and immature embryos were analyzed after bombardment with different types (version) of GFP genes and driven by different constitutive promoters. Some of the GFP genes used were targeted to specific organelles within the cell, namely mitochondria, plastid and endoplasmic reticulum (ER). For transformation using non-targeted GFP genes, plasmid p35S.TYG.GFP showed the highest number of green fluorescent spots, followed by pHBT-sGFPS65T and pGEM.Ubi-sGFPS65T. The results demonstrated that the sGFPS65T gene version is the most active in oil palm. When organelle targeted GFP genes were transformed, localization of the genes into specific organelle was clearly demonstrated using laser scanning confocal microscopy. Using the organelle targeted GFP genes; the sGFPS65T gene version was again demonstrated to be the most active in oil palm cells. Long term transient expression of the GFP genes was also evaluated and is discussed.

PLoS ONE, 2014

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.

Transformation vector construction is one of the important disciplines for plant genetic transformation studies. A series of vector consisting of hygromycin phosphotransferase (hpt) gene as the selective marker and green fluorescent protein (GFP) as the visual reporter gene, under the control of double cauliflower mosaic virus 35S (2XCaMV35S) promoter has been engineered for transformation into oil palm cells. These genes were cloned into different types of cloning and expression vectors. The cloning was carried out by using restriction enzyme digestion and ligation method. Five intermediate vectors have been created for insertion of 2XCaMV35S-HPT-35ST and 2XCaMV35S-GFP-35ST into modified pBINPLUS backbone vector for particle bombardment and Agrobacterium-based transformation protocols. All vectors were sequenced to confirm the integrity of DNA region. The vectors were later transformed into oil palm embryogenic calli using biolistic device. The viability of the vectors was initially evaluated by transient GFP fluorescence expression observed under fluorescence microscope. It was demonstrated that the 2XCaMV35S promoter was able to drive the expression of gfp as gene in oil palm calli.

Nature Precedings

The African oil palm, Elaeis guineensis is native to Africa, occurring between Angola and Gambia, while the American oil palm Elaeis oleifera is native to tropical Central America and South America. The African oil palm, Elaeis guineensis is placed in the Arecaceae family along with coconut and date palms. It has been elucidated the genetics of three naturally occurring fruit types includes dura, tenera, and pisifera. The dura (sh + sh +) is for the presence of a relatively thick shell (2-8 mm). The tenera (sh + sh-) with a relatively thin shell (0.5-4 mm). In comparison to tenera and dura, the pisifera (shsh-) is known for the absence of the shell (Hardon et al., 1985). The popular cultivar is tenera which yield more oil concentration. Oil palm is a monocotyledonous plant that has the average height is about 20-30 feet in cultivation. The oil palm leaves are up to 25 feet in length containing leaflets around 200-300 per leaf. Leaf bases are persistent for years, and prominent leaf scars are arranged spirally on the trunk of mature palms where bases have fallen. A young tree produces about 30 leaves a year, the trees over ten years can produce 20 leaves per year. Oil palm is monoecious which enables cross-pollination because male and female inflorescence are produced from axillary buds separately to each other. The flowers are produced in dense clusters; each individual flower is small, with three sepals and three petals. Fruits are born on secondary branches, it has ovoid shape and elongate 2-5 cm in length, the weight varies between 5-20 g. Fruits are tightly attached to fruit bunches, the number of fruit per bunch is between 50 to 100 in young palms and up to 1000-3000 in older ones. Ripe bunches weigh from 20 to 50 kg (Hardon et al., 1985). Oil palm takes roughly 7 years to bear fruits. Oil palm fruit takes six months to mature; when fruits ripen, the color turns from black to orange, the degree of darkness depending on light exposure and cultivar. Oil palm fruit includes four parts, the outer layer is the 2 pericarp that has a tough surface to protect the inner ones. The next layer is mesocarp that is rich in oil from which the industrial products are manufactured, used mainly for producing cooking oil. The endocarp or the shell is next, it usually appears as a black firm layer that covers the last part-kernel, the oil extracted from this part is normally for processing industrial foods. The annual harvested production for each hectare of oil palm is about 10 tonnes of fruits, which yields 3,000 kg of pericarp oil, and 750 kg of seed kernels, which yield 250 kg of extracted kernel oil as well as 500 kg of kernel meal. The meal is used for livestock breeding. Some varieties have even higher productivities which can be used for producing the vegetable oil used in biodiesel. 1.1.2 The use of oil palm Oil palm has many applications in the industrial production, edible palm oil-based fats are primary element used to make processed food products such as cooking oil, shortening, margarines, ice creams and cookies. Palm oil specialty fats are also applied as a cocoa butter substitute in chocolates. Oil palm is now edible and has surpassed soybean oil as the most popular vegetable oil in the world with higher production (Table 1.1). Other products produced from oil palm are soap, washing powders and cosmetics, medicines and future diesel fuel substitute. These aforementioned products are produced from the crude palm oil derived from the mesocarp part. The kernel meal after extracting kernel oil is the secondary product is mainly used for animal feed. The relative high level of palmitate (about 44%) provide a stable level of sustainment that is suitable for high temperature frying application (Broun et al., 1999). The approximately 40% oleate and 10% linoleate of palm oil has been shown to lower total serum cholesterol, functioning in movement of cholesterol within the blood stream by reducing the level of the lowdensity lipoprotein cholesterol (Sundram et al., 2003). Additionally, the high level of the high-density lipoprotein and the removal of cholesterol from the bloodstream has been proven to protect against heart diseases (Sundram, 1997; Pedersen et al., 2005). Table 1.1 Oil productivity of major oil crops. Oil crop Oil production

International Journal of Agricultural Technology, 2012

The influence of density of Agrobacterium, inoculation and co-cultivation period on gene transformation in embryogenic callus of oil palm was investigated. Agrobacterium strain AGL1 containing plasmid pCAMBIA1304 which carring gus and hpt as screenable and selectable marker genes, respectively, was used for transformation. The results showed that embryogenic callus at 4 weeks inoculated in Agrobacterium suspension at density of 0.8 (OD600) for 6 hours and co-cultivated on solid MS medium with 200 μM acetosyringon in the dark for 3 days, followed by culturing on selective medium for 2 weeks gave the highest transient expression of gus gene at 100%. The hygromycin-resistant embryogenic callus was obtained at 63.89% and developed to form somatic embryos at 4 embryos/culture after 8 weeks of culture on selection medium. Polymerase chain reaction (PCR) analyse confirmed the presence of gus gene at size of 441 bps and hpt gene at size of 800 bps.