Soybean protoplasts from a number of commercially important cultivars have been genetically engineered by way of electroporation using chimeric genes coding for resistance to the aminoglycoside antibiotics kanamycin and G418. Effective electroporation conditions were determined by monitoring transient expression from aminoglycoside 3'-phosphotransferase II (APHII) expression plasmids. Electroporation of protoplasts with a chimeric APHII gene and subsequent selection on media supplemented with kanamycin resulted in the recovery of calli resistant to the antibiotic. Enzyme assays for APHII activity and Southern blot hybridization confirmed the expression of the foreign DNA and its stable integration into the soybean genome. Root formation was induced from transformed calli, and these roots maintained expression of the APHII gene.Soybean (Glycine max) is one of the world's most important agronomic crops. Accordingly, a great deal of effort has been directed towards its genetic improvement by both conventional breeding techniques and genetic engineering approaches. Successful application of standard genetic engineering procedures to soybean has been limited by the lack of an efficient transformation system and the inability to regenerate transformed tissues. Oncogenic transformation of soybean by virulent Agrobacterium strains and the axenic culture of excised tumors on hormone-free media have been reported (1-5), but disarmed Ti vectors have not yet been used successfully. Agrobacterium strains have been isolated that show increased virulence toward soybean and other plants that are not particularly susceptible to crown gall infection, and "disarmed" hosts for Agrobacterium vector systems have been developed from these strains (6). However, even with these strains, the transformation frequency is very low relative to that obtained in alternative plant hosts such as tobacco and petunia. Electroporation has been shown to be an effective technique for the transformation of mammalian cells (7). Fromm et al. (8) have shown that with appropriate modification the technique is also applicable to plant protoplasts, and they have achieved the stable transformation of maize tissue cultures by this method (9). These results, together with our own experience with electroporation of tobacco, prompted us to investigate this method as a means of achieving the transformation of soybean. MATERIALS AND METHODSPlasmids. pCMC1021 was constructed from a fragment of pBR322 [nucleotides 2522-4361 (10)] containing the replication origin and the 8-lactamase gene, a chimeric gene consisting of 5' and 3' regulatory regions from the nopaline synthase gene of pTiT37 [nucleotides -265 to +36 and + 1298 to +1550, respectively (11)], and the aminoglycoside 3'-phosphotransferase II (APHII; kanamycin kinase, EC 2.7.1.95) coding region from TnS [nucleotides 1541-2517 (12)]. The coding region deletes an AUG triplet present in the TnS sequence upstream and out-of-frame with respect to the initiator AUG (13). The junctions between the pBR32...