Transient gene expression in cassava protoplasts

The aim of the project is the development and improvement of methodologies for gene transfer in cassava. Isolated protoplasts provide a convenient system for studying the parameters influencing the transformation of protoplasts by direct DNA uptake. Transient expression studies have utilized a plasmid pAL carrying the beta-glucoronidase gene under control of 35S-CaMV promoter and nos polyadenilation region, following electroporation-mediated plasmid uptake. Protoplasts were isolated from cassava leaves (var. MCol22) growing in vitro, resuspended in electroporation buffer (20 mM MES, pH 5.8, 10 mM CaCl2) and 20 ug ml-1 plasmid. A viability curve of the protoplasts was initially established utilizing different electroporation parameters. The optimum electroporation parameter gave an electric field of 715 V.cm-1 and a time constant =12.8 msec. The expression of the gene product was analyzed by a qualitative and quantitative beta-glucuronidase enzyme assay. The results obtained from the foundation to study direct gene transfer in cassava. In addition, may allow a rapid method to evaluate the functional expression of tissue-specific promoters. Nevertheless, experiments have been carried out utilizing particle bombardment using tissues with regeneration capability. The production of transgenic plants should facilitate, in the future, studies to the introduction and expression of important agronomically traits in cassava.