Transformation of sweet potato tissues with green-fluorescent protein gene

The expression of the green-fluorescent protein (GF?), gene from Aequorea v ictoria (jellyfish) was analyzed by transient and stable expression in, swe et potato Ipomoea batatas L. (Lam.) cv. Beauregard tissues by electroporati on. and particle bombardment. Leaf and petiole segments from in vitro-raise d young plantlets were used for protoplast isolation and electroporation. E mbryogenic callus was also produced from leaf segments for particle bombard ment experiments. A buffer solution containing, 1 x 106 protoplasts ml(-1) was mixed with plasmid DNA containing the GFP gene, and electroporated at 3 75 V cm(-1). Approximately 25-30% of electroporated mesophyll cell protopla sts subsequently cultured in KM8P medium regenerated cell walls after 48, h . Of these, 3% emitted bright green fluorescence when exposed to, UV-blue l ight at 395 nm. Transformed cells continued to, grow after embedding in KM8 P medium solidified with 1.2% SeaPlaque agarose, Stable expression of GFP w as observed after 4 wk of culture in approximately 1.0% of the initial GFP positive cells (27.5, GFP positive micro calluses out of 3024 cells which t ransiently expressed GFP 48 h after electroporation). In a separate experim ent, 600-700 bright green spots were observed per plate 48 h after bombardi ng leaf segments or embryogenic: callus. In bombarded cultures, several sta ble GFP-expressing sectors were observed in leaf-derived embryogenic callus grown without selection for 4 wk. These results show that GFP gene express ion can occur in various sweet potato, tissues, and that it may be a useful screenable marker to, improve transformation efficiency and obtain transge nic sweet potato plants.