Читать книгу Biotechnology of Fruit and Nut Crops - Группа авторов - Страница 148

Izawati et al. (2012) reported the regeneration of transgenic plantlets after Agrobacterium tumefaciens-mediated transformation of embryogenic cultures derived from young leaves. Transformation involved Agrobacterium strain LBA4404, harbouring the plasmid pUBA, which carries a selectable marker gene (bar) for resistance to the herbicide Basta 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 biolistics were applied. Transgenic oil palm cells were selected and regenerated on a medium containing herbicide Basta. Molecular analyses revealed the presence and integration of the introduced bar gene into the genome of the transformants.

Genetic engineering remains a major challenge in oil palm because particle bombardment (Parveez, 2008; Parveez and Bahariah, 2012) and Agrobacterium-mediated transformation (Yenchon and Te-chato, 2012) are labourious and/or inefficient with this species, often producing chimeric plants and escapes. Protoplasts are useful as targets 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 recovery of transgenic oil palm plants. Masani et al. (2014) achieved the regeneration of healthy and fertile transgenic oil palms from protoplasts. These authors focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating: (i) optimal heat shock conditions; (ii) concentrations of DNA, polyethylene glycol (PEG) and MgCl₂; and (iii) the transfection procedure. Results indicated that 100 ng/μl was the optimal concentration of microinjected DNA. The effect of heat shock treatment was investigated by incubating the protoplasts at 45°C for 5 min and then cooling on ice for 1 min before adding 50 μg of CFDV-hrGFP plasmid DNA, incubating for 10 min as above and then adding 25% (w/v) PEG in 50 mM MgCl₂. This treatment increased the transfection efficiency even further to 4.76% indicating that a heat shock significantly improves DNA uptake. No difference in terms of green fluorescent protein (GFP) fluorescence could be found regardless of the PEG concentration, suggesting that PEG does not affect hrGFP gene expression but may instead affect the viability of the oil palm protoplasts at concentrations higher than 25%. The presence of 10 mM MgCl₂ increased the transfection efficiency by fourfold to 0.39% compared to a PEG solution lacking magnesium but higher concentrations were even more beneficial, and the greatest efficiency (2.5%) was achieved in the presence of 50 mM MgCl₂. The authors also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalluses that expressed GFP.