Direct selection of cybrids by streptomycin and valine resistance in tobacco

Direct selection of cybrids by simultaneous selection for "donor" chloroplasts and for the "recipient" nuclei is described. Mesophyll protoplasts of two tobacco (Nicotiana tabacum) mutants, SR1 (streptomycin resistant) and Val(r)-2 (valine resistant), were fused by polyethylene glycol treatment. Streptomycin resistance in the SR1 mutant is a maternally inherited chloroplast trait while valine resistance is a Mendelian (nuclear) digenic recessive character. The fused protoplast population was cultured and colonies were selected for resistance to valine (1 mM) and streptomycin (343 μM). The efficiency of selection has been confirmed in three clones by demonstrating seed transmission of both streptomycin and valine resistances. In one subclone both streptomycin resistant and sensitive plants were obtained indicating that the streptomycin sensitive chloroplasts had not been totally eliminated by growth on the selective medium.