Abstract: Protoplasts were prepared from 2 days old subcultures of soybean (Glycine max (L.) Merr.) and fragments of young leaves of tobacco (Nicotiana tabacum var. “Xanthi”) according to the methods of Kao. Protoplasts were fused and single fusion products were cultured in Cuprak dishes as previously described. Fusion products were fixed and embedded in plastic by reported methods for electron microscope study. Isoenzyme studies were carried out according to described methods. Proteins were electrophoresed on 5% polyacrylamide gels and stained. Fusion products were easily identified on the basis of the presence of both tobacco chloroplasts and soybean leucoplasts (Fig. 1). The chloroplasts contained typical grana and stroma lamellae; leucoplasts were characterized by numerous starch granules and a paucity of internal lamellae. After 15 hours in culture, thorough mixing of cytoplasm had occurred as evidenced by the distribution of plastids. Fusion of interphase nuclei was not observed in any of the fusion products. Premitotic nuclear fusions which have been reported previously may signify unhealthy fusion products. Fusion products underwent their first cell division within 2–3 days in culture; divi- ding nuclei contained complete sets of both tobacco and soybean chromosomes. During subsequent divisions, hybrids gradually lost some tobacco chromosomes. By 4.5 days, small clusters of hybrid cells were evident. The chloroplasts of such hybrid cells exhibited unusual shapes, possibly as a result of starch accumulation (Fig. 2b, 2c). The leucoplasts remained unchanged. Within 2 weeks, hybrid clusters contained 100–200 cells. Very few chloroplasts were detected in these cells by electron microscopy. The chloroplasts present were highly modified. Typically, these plastids were characterized by enlarged grana and elongated parallel stacks of stroma lamellae. Similar changes in plastid morphology were observed in pea-soybean fusion products cultured for 1 week. It is not possible to determine from the present study whether chloroplasts were being diluted during cell proliferation or whether they were dedifferentiating. Previous ultrastructural research suggests that dedifferentiation of chloroplasts occurs in fusions involving similar species while chloroplasts degeneration is more likely in fusions of widely separated species . Biochemical evidence from studies of the electrophoretic mobility of the plastid-encoded large subunit of ribulose-1, 5-bisphosphate carboxylase and the endonuclease restriction patterns of plastid DNA indicate that plastids may either assort randomly or both plastid types may coexist in cells of regenerated hybrid plants. Chloroplasts were not detected in hybrids cultured for prolonged periods. The leucoplasts in these cells were indistinguishable from leucoplasts of parental soybean cells. Leucoplasts were not diluted during cell division and their numbers were likely maintained by plastid division. Over 20 hybrid cell lines were established and cultured for 7–9 months. Chromosome analysis revealed that many lines including the one illustrated in Fig. 4 retained over one half of the tobacco chromosomes in addition to the full soybean chromosome complement . Zymograms from this same cell line are presented in Fig. 5. The electrophoretic patterns for both dehydrogenases clearly demonstrate that hybridization has been achieved. The shikimate dehydrogenase (SDH) zymogram for the hybrid shows that the broad slow- moving band from soybean and the 2 distinctive fast-moving bands found in Nicotiana are all present in the hybrid. Similarly, for 6-phosphogluconate dehydrogenase (6-PGDH), the hybrid contained the bands from soybean and the 3 slower-moving bands from Nicotiana as well as one of the 2 fast-moving bands found in the latter. This study demonstrates the usefulness of both electron microscopy and isozyme analysis for examining hybrid cells derived from plant protoplast fusion. During the early stages of hybrid culture when small sample size precludes isozyme analysis, ultrastructure studies permit the identification of hybrid cells, after prolonged culture, the isozyme technique is a much more sensitive measure of hybridization than is electron microscopy.