Tobacco protoplast transformation and use for functional analysis of newly isolated genes and gene constructs

This chapter discusses the tobacco protoplast transformation and use for the functional analysis of newly isolated genes and gene constructs. It has been known for about two decades that, under certain conditions, plant protoplasts can take up viruses, microorganisms, and nucleic acids. After the development of chimeric genes, direct gene transfer (DGT) and the generation of transgenic plants were reported. Since then, numerous reports of DGT into plant protoplasts have been published. DGT is not restricted to certain plant species, but the breadth of its application depends on the ability of protoplasts to divide subsequently and regenerate. DNA uptake methods are a valuable tool for the rapid analysis of promoter/reporter gene constructs and can be used for studying promoter or other regulatory elements that influence transcription. If protoplast regeneration is possible, the expression of the transferred DNA can also be analyzed at the level of the regenerated callus or plant. © 1995, Academic Press, Inc.