Protoplasts in organelle research: Transfer and transformation of plastids

Protoplasts, because they lack the wall of a typical higher plant cell, offer unique opportunities for experimental manipulation of their organellar constituents. Here, we report on modification of the organellar content of Nicotiana tabacum protoplasts by microfusion-induced transfer of defined numbers of chloroplasts into albino recipient cells. A single chloroplast is found to be sufficient for establishing a new plastid population in the progeny of the recipient cell. The frequency of green or variegated regenerants is shown to be genotype dependent. It can be drastically increased by using selection pressure for the transferred organelle. We also report on transient expression of plastid specific reporter gene constructs in plastids after PEG-mediated direct gene transfer into Nicotiana plumbaginifolia protoplasts. The expression is shown to be localized in the plastids by determining gene expression in isolated chloroplasts under conditins which completely remove cytoplasmic enzyme activity derived from a nuclear reporter gene construct. These data demonstrate for the first time that functional DNA, introduced into the cytoplasm by direct gene transfer, enters the organellar compartment and is expressed.