Transfer of defined numbers of chloroplasts into albino protoplasts using an improved subprotoplast/protoplast microfusion procedure: transfer of only two chloroplasts leads to variegated progeny.

A procedure is described by which it is possible to perform controlled microfusion of microscopically selected protoplast fusion partners with high efficiencies. The procedure is applied to fusion of Nicotiana tabacum (line 92V37. N. undulata cytoplasm) plastid albino protoplasts as a recipient and spontaneously formed subprotoplasts of green N. tabacum (line SR1) as donor. Products of individual electrofusion events are cloned via single cell nurse culture and the derived cell lines are analysed for the occurrence of variegated or green regenerating shoots, which are indicative of the establishment of the transferred organelles in the cell progeny. The plastid population in green regenerants recovered after the transfer of only two chloroplasts was demonstrated to have originated from the donor subprotoplast organelles by restriction analysis of total DNA using a plastome-specific probe.