An improved procedure for protoplast microelectrofusion and culture of Nicotiana tabacum intraspecific somatic hybrids: plant regeneration and initial proofs on organelle segregation

Somatic hybrids were produced between haploid Nicotiana tabacum L. cv. Petite Havana (wild type) and haploid streptomycin resistant (SR1) mutant by an improved version of microelectrofusion of preselected pairs of protoplasts and the culture of fusion products in a nurse culture. Resistance of diploid plants regenerated from 20 somatic hybrid clones was tested at low concentration of streptomycin in the light as well as at high concentrations of streptomycin in the dark. In two independent hybrid lines, plants resistant in the light but sensitive in the dark were found. The existence of this plant type indicates a segregation of chloroplasts and mitocondria in somatic hybrid clones. It is suggested that microelectrofusion of preselected pairs of protoplasts combined with a reliable nurse culture might be a good technique for controlled somatic hybridization, cell reconstitution and partial gene transfer to different plant species. It might also be used to follow and analyse organelle segregation in somatic hybrid clones. The possibility that mitochondria might be resistant to streptomycin in the SR1 mutant is also discussed.