Direct DNA transfer to protoplasts with and without electroporation

Abstract

The introduction of ‘naked’ DNA into plant protoplasts, in comparison to transformation methods used for bacterial, yeast and animal cells, has been a relatively recent development. The first conclusive demonstrations of uptake and integration of DNA into plant protoplasts were those of Davey et al. [1], Draper et al. [2] and Krens et al. [3] in which isolated Ti plasmid from Agrobacterium tumefaciens was applied to plant protoplasts in the presence of poly-1-ornithine or polyethylene glycol/Ca^{2 +}. The presence of the Ti DNA in the plant genome was demonstrated both by the phenotype of hormone auxotrophic growth, production of the expected opine and by Southern blot analysis of DNA from the transformants. The DNA integrated into the genome appeared to be a random assortment of DNA derived from the Ti plasmid. The subsequent development of markers allowing positive selection in plant cells (i.e. antibiotic resistance markers) led to the development of a much simplified protoplast transformation system. Pazskowski et al. [4] constructed a simple plasmid based on pUC8 containing a selectable marker, the kanamycin resistance gene from the trans-poson Tn5 with expression signals from gene VI of the dsDNA virus cauliflower mosaic virus (CaMV). Using this plasmid (pABDl) and an uptake method derived from that of Krens et al [3], they were able to demonstrate the uptake, integration and expression of the resistance marker in protoplasts of Nicotiana tabacum.