Method for the generation of a monoclonal plant cell line

A method for the generation of a monoclonal plant cell line from a heterogeneous population of plant cells, comprising the following steps:

• (a) providing a heterogeneous population of plant cells;
• (b) preparing a heterogeneous population of protoplasts from the heterogeneous population of plant cells, wherein at least one of the protoplasts from the prepared heterogeneous population of protoplasts is transformed with at least one expression vector encoding at least one fluorescent protein and a resistance against at least one selection agent;
• (c) initiating cell wall regeneration in said heterogeneous population of protoplasts for fluorescence activated cell sorting (FACS);
• (d) providing a cell deposition device containing feeder cells in liquid medium containing wells;
• (e) sorting by FACS the prepared at least one protoplast from the heterogeneous population of protoplasts in which the cell wall regeneration has been initiated, wherein said at least one protoplast is highly fluorescent;
• (f) separating the sorted at least one protoplast into at least one different liquid medium-containing well containing the feeder cells for monoclonal plant cell generation such that said at least one well for monoclonal plant cell line generation contains a single prepared protoplast permitted to directly contact the feeder cells;
• (g) regenerating the single separated protoplast from said at least one well into a microcolony by co-cultivating the separated protoplast in the same well as the feeder cells;
• and
• (h) removing the microcolony from the feeder cells and cultivating the microcolony in the presence of said at least one selection agent against which said at least one protoplast is transformed with said at least one expression vector encoding a resistance until a monoclonal plant cell line from said at least one protoplast is established.