Production of herbicide-resistant Highbush blueberry 'Legacy' by Agrobacterium-mediated transformation of the bar gene

The chimeric bialaphos resistance (bar) gene driven by the nos promoter was introduced into highbush blueberry, Vaccinium corymbosum L. 'Legacy', using Agrobacterium tumefaciens-mediated transformation (Song and Sink, 2004). Twenty-one out of 300 leaf explants inoculated with A. tumefaciens strain EHA105:pGTV-BAR regenerated shoots after selection on modified Woody Plant Medium + 1.0 mg L-1 TDZ + 0.5 mg L(-1)NAA + 250 mg L-1 timentin + 0.1 mg L-1 glufosinate ammonium (GS). Cloned shoots of randomly selected and PCR-positive transgenic lines L18, L21, L22 and L25 showed resistance to a higher level of GS at 5.0 mg L-1 during chlorophenol red assays and were studied further. The integration and expression of the bar gene in these 4 lines was confirmed by Southern hybridization and by phosphinothricin acetyltransferase (PAT) assays. L18, L21 and L22 lines each had a single copy and L25 had three copies of the bar gene. Herbicide glufosinate at four levels (GS in mg L-1: 0, 750, 1500 and 3000) was applied using a track sprayer on 8-month old plants growing in planting medium. Evaluations done after 2 weeks indicated that at the high-dose of GS (3000 mg L-1), 4 times the standard level for field application, the percents of leaves with no injury symptoms were 70%, 77%, 97% and 71%, respectively, for L21, L22, L18, and L25. Only 7% of the leaves on non-transgenic plants had no injury and 66% abscised. These results indicate that the bar gene may serve both as a selectable marker and/or for herbicide-resistance for blueberry as currently being further evaluated in a field trial.