Protoplast Isolation and Fusion
Plant protoplasts provide a unique single-cell system to underpin several aspects of modern biotechnology. Major advances in genomics, proteomics, and metabolomics have stimulated renewed interest in this osmotically fragile wall-less cells. Reliable procedures are available to isolate and culture protoplasts from a range of plants, including both monocotyledonous and dicotyledonous crops. Importantly, novel approaches to maximize the efficiency of protoplast-to-plant systems include techniques already well established for animal and microbial cells, such as electrostimulation and exposure of protoplasts to surfactants and respiratory gas carriers, especially perfluorochemicals and hemoglobin. However, despite at least four decades of concerted effort and technology transfer between laboratories worldwide, many species still remain recalcitrant in culture. In the context of plant genetic manipulation, somatic hybridization by protoplast fusion enables nuclear and cytoplasmic genomes to be combined, fully or partially, at the interspecific and intergeneric levels to circumvent naturally occurring sexual incompatibility barriers. Uptake of isolated DNA into protoplasts provides the basis for transient and stable nuclear transformation, and also organelle transformation to generate transplastomic plants. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, synthesis of pharmaceutical products, and toxicological assessments.