REGENERATION OF TRANSFORMED SHOOTS FROM ELECTROPORATED SOYBEAN (GLYCINE-MAX (L) MERR) PROTOPLASTS

Stable transformation of soybean (Glycine max (L.) Merr.) protoplasts isolated from immature cotyledons was achieved following electroporation with plasmid DNA carrying chimeric genes encoding beta-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transformed colonies were stringently selected by growing 15-day-old protoplast-derived cells in the presence of 40-mu-g/ml of hygromycin-B for 6 weeks. Over 93% of the resistant cells and colonies exhibited GUS activity, indicating that the two marker genes borne on a single plasmid were co-introduced and co-expressed at a very high frequency. This transformation procedure reproducibly yields transformants at frequencies of 2.9-6.8 x 10(-4) (based on the number of protoplasts electroporated) or 23.0% (based on the number of control microcalli formed) counted after 6 weeks of selection. After repeated subculturing on regeneration medium, shoots were induced from 8.0% of the transformed calli. Southern hybridization confirmed the presence of both the GUS and hygromycin genes in the transformed calli and shoots.