Abstract:Isolated pollen protoplasts of Nicotiana tabacum L. N364 Km+ were fused with mesophyll protoplasts of N. rustica L. using PEG-high Ca/pH method. The cells resulted from fusion between immature (early-middle bicellular) pollen protoplasts and mesophyll protoplasts could divide to produce microcalli and regenerated plantlets when cultured in a selection KM8p medium containing 50 g/L kanamycin. Four plantlets were regenerated. The isoenzyme patterns of leaf peroxidases of these plantlets had bands characteristic of both parents. Root-tip squash showed that the gameto-somatic hybrids had the expected triploid chromosome number. Aside from these kanamycin-selected plantlets, six of the twenty-one plantlets that had not undergone selection were also evidenced to be gameto-somatic hybrids.