Abstract
BackgroundGenetic 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 FindingsWe 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/SignificanceWe 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.
Highlights
The oil palm genetic engineering program was initiated by the Malaysian Palm Oil Board (MPOB), known as the Palm Oil Research Institute of Malaysia (PORIM), in the early 1990s [1]
We investigated the impact of Mg2+ ions on transfection efficiency by incubating oil palm protoplasts as above for 10 min in the presence of 10 mg of CFDV-hrGFP plasmid DNA mixed with 40% (w/v) polyethylene glycol (PEG) dissolved in Rinse solution, but this time we varied the concentration of Mg2+ ions by preparing solutions containing 10 mM (Figure 2A), 25 mM (Figure 2B), 50 mM (Figure 2C) and 100 mM MgCl2 (Figure 2D)
Genetic engineering in oil palm is challenging because the standard transformation approaches based on Agrobacterium and particle bombardment are laborious and inefficient, generating a large number of chimeric plants
Summary
The main objectives of this program are to produce transgenic oil palm (Elaeis guineensis) with a higher content of oleic acid, modified oil quality (e.g. a higher content of stearic acid), and the ability to produce value-added oils such as palmitoleic and ricinoleic acid, as well as novel products such a biodegradable plastics. A biolistic protocol for the production of glufosinate-resistant transgenic oil palm has been developed [4], and in light of its success thousands of embryogenic calli have been bombarded with genes involved in fatty acid biosynthesis to increase the accumulation of oleic acid [5,6,7], stearic acid [8], polyhydroxybutyrate (PHB) and polyhydroxyvalerate (PHBV) [9,10,11]. 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. Novel approaches for the transformation of oil palm protoplasts could offer a new and efficient strategy for the development of transgenic oil palm plants
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