Selection of hybrid plants obtained by electrofusion of vacuolated x evacuolated plant protoplasts in hypo-osmolar solution.

Vacuolated and evacuolated tobacco mesophyll protoplasts were electrically fused in hypo-osmolar media by using an alternating field of modulated amplitude for alignment. The vacuolated fusion partner was isolated from Nicotiana tabaccum L. cv Xanthi and the evacuolated one from the streptomycin-resistant strain Nicotiana tabaccum L. cv Petit Havana SR1. The field and osmolarity conditions used ensured relatively high yields of heterologous fusion products despite the differences in density and size of the parental cells. After removal of the evacuolated, streptomycin-resistant fused and unfused protoplasts by flotation of vacuole-containing cells on iso-osmolar sucrose medium, the cybrids and hybrids were cultured in 25 microliters drops of agarose. During the first 5 weeks the non-fused Xanthi-protoplasts were used as a nurse culture. After addition of streptomycin to the growth media, cybrids and hybrids were successfully selected whereas fused and unfused vacuole-containing protoplasts died within 6 days. Only the streptomycin-resistant cybrids and hybrids developed into whole plants. On average a yield of 0.025% of streptomycin-resistant plants (referred to the total number of parental cells) was obtained. Polyacrylamide gel electrophoresis of leaf extracts of these plants showed that at least 50% of the streptomycin-resistant plants had a hybrid-esterase isoenzyme pattern. The protocol can be generalised by fusion of iodoacetamide-inactivated vacuolated protoplasts with meristematic (or evacuolized) protoplasts carrying no genetic marker. Use of evacolated protoplasts for electrofusion with vacuole-containing protoplasts therefore offers a way of overcoming the lack of suitable genetic markers for hybrid selection.