Transformation of sweet potato tissues with green-fluorescent protein gene

Abstract

The expression of the green-fluorescent protein (GFP) gene from Aequorea victoria (jellyfish) was analyzed by transient and stable expression in sweet potato Ipomoea batatas L. (Lam.) cv. Beauregard tissues by electroporation and particle bombardment. Leaf and petiole segments from in vitro-raised young plantlets were used for protoplast isolation and electroporation. Embryogenic callus was also produced from leaf segments for particle bombardment experiments. A buffer solution containing 1 Â 10 6 protoplasts ml 21 was mixed with plasmid DNA containing the GFP gene, and electroporated at 375 V cm 21 . Approximately 25±30% of electroporated mesophyll cell protoplasts subsequently cultured in KM8P medium regenerated cell walls after 48 h. Of these, 3% emitted bright green fluorescence when exposed to UV-blue light at 395 nm. Transformed cells continued to grow after embedding in KM8P medium solidified with 1.2% SeaPlaque agarose. Stable expression of GFP was 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 transiently expressed GFP 48 h after electroporation). In a separate experiment, 600±700 bright green spots were observed per plate 48 h after bombarding leaf segments or embryogenic callus. In bombarded cultures, several stable GFP-expressing sectors were observed in leafderived embryogenic callus grown without selection for 4 wk. These results show that GFP gene expression can occur in various sweet potato tissues, and that it may be a useful screenable marker to improve transformation efficiency and obtain transgenic sweet potato plants.

Plant protoplast technology: Current status

“…A ma jor lim i tation of DNA up take into proto plasts is the low frequency of sta ble trans for ma tion, usu ally about 1 in 10 4 cells. Be cause of this fre quency, the ma jor exploi ta tion of DNA up take into iso lated proto plasts is cur rently in short-term ex per i ments of tran sient gene ex pres sion to eval u ate the per for mance of new gene con structs, fre quently in volv ing the beta-glucuronidase (gus) or green flu o res cent protein (gfp) genes as re port ers (Winfield et al 2001, Sheen 2001.…”

Regeneration of transformed verbena (Verbena × hybrida) by Agrobacterium tumefaciens

“…For this purpose, the green fluorescent protein (GFP) has been widely used for transformation experiments, and various GFP mutants have been developed (Stewart 2001). As GFP can visualize and simplify the tracking of transgenic events, transformation by the GFP gene has recently been applied to recalcitrant or economically important species to establish a new and efficient transformation system (Ghorbel et al 1999;Iocco et al 2001;Li et al 2001;Winfield et al 2001) or to study trans-gene regulation (Chytilova et al 1999;Weld et al 2001;Wymer et al 2001). In the investigation reported here, we used the sGFP (S65T) gene, one of the GFP mutants (Chiu et al 1996;Niwa et al 1999) as a marker gene to trace and evaluate the delivery of a transgene and successfully produced the first genetically modified verbena plants by Agrobacterium-mediated transformation.…”