Development of transformation vectors for the production of potentially high oleate transgenic oil palm

The main target of Malaysian Palm Oil Board (MPOB) genetic engineering programme is to produce high oleate transgenic palms. The availability of effective transformation vector is one of the pre-requisites for genetic manipulation of oil palm through recombinant DNA technology. Here, we describe the construction of a series of transformation vectors that have a maize ubiquitin promoter (UbiPro)-driven bar gene for selection of transformants on herbicide (Basta or Bialaphos), and mesocarp-specific promoter (MSP1) for expression of the transgenes [antisense palmitoyl-ACP-thioesterase (PAT) and sense β-ketoacyl-ACP-synthase II (KASII) and sense D9-stearoyl-ACP-desaturase (SAD)] potentially responsible for high oleate content in oil palm mesocarp. The transformation vectors constructed in this study are suitable for use in both particle bombardment (biolistic) and Agrobacterium-based transformation protocols

Determining the optimal concentration of mannose as an effective selection agent for transformed oil palm cells using the phosphomannose isomerase (pmi) gene as a positive selectable marker

The elimination of antibiotic or herbicide resistance gene usage in genetically modified plants is being encouraged due to public concern. In response to this, alternative selection systems for the recovery of transgenic oil palm were developed using positive selectable markers. To establish a selection system that utilises the phosphomannose isomerase (pmi) gene for oil palm transformation, we first determined the optimal mannose concentration for selecting the transformed cells. Non-transformed embryogenic calli were cultured on media containing various combinations and concentrations of mannose and a usable source of carbon, i.e. sucrose, ranging in content from 0 to 30 g litre . Sucrose is often used as a carbon source in plant tissue culture media. The embryogenic calli were subcultured onto similar fresh media every four weeks, and growth was recorded monthly up to five months. From the 10 combinations of mannose and sucrose evaluated, mannose:sucrose at 30:0 g litre was shown to be the most effective for selection because at this concentration the least plant grozvth was demonstrated for non-transformed embryogenic calli. We will thereafter use this particular concentration of mannose to select for oil palm embryogenic calli transformed with the pmi gene using biolistic bombardment

Optimization and transformation of garden balsam, impatiens balsamina, mediated by microprojectile bombardment

In this study, a transformation system was developed by initially optimizing the microprojectile bombardment parameters for cotyledonous explants. The parameters optimized were combination of distance from stopping screen to target tissue and helium pressure, number of bombardment, preculture duration prior to bombardment, DNA amount, osmoticum treatment and post-bombardment incubation time. Determination of minimal inhibitory concentration for efficient selection of transformants was also carried out for hygromycin. Different concentrations of hygromycin (25. 50, 75 and 100 mg L-1) were tested against different ages of explants (0, 1, 3 and 5 weeks) for efficient selection of transformants. Using the optimized parameters, transformation of cotyledons was carried out followed by selection on 75 mg L-1 hygromycin at different weeks of post-bombardment. Transgenic plants were successfully regenerated from bombarded explants and confirmed via histochemical GUS assay and PCR analysis