Inhibition of cell wall synthesis improves flow cytometric sorting of potato heterofusions resulting in hybrid plants

Potato heterofusions obtained after electrofusion of diploid lines of Solanum tuberosum and Solanum phureja were sorted using a FACS IV flow sorter. Adjustments of this apparatus include a motor-driven syringe for injection of the protoplasts and heterofusions and use of a 200 μm nozzle orifice. Recovery of intact heterofusions was improved by adding after fusion an equal volume of culture medium containing 2,6-dichlorobenzonitrile (DB) (20 μg/ml) to the fusion mixture. Sorting was performed after 3–5 h. Plantlets regenerated from the heterokaryons were analyzed for their ploidy level and hybrid character by flow cytometric measurements of DNA contents: from 39 plant regenerating calli, 14 were tetraploid. Six tetraploid plants have been shown by Giemsa C-banding of their chromosomes to be hybrids. It is concluded that flow sorting of heterofusions compared to their collection using a micromanipulator may result in isolation of a higher fraction of one to one fusions rather than multifusions.