Production of Marker-Free Transgenic Lettuce with Resistance to Mirafiori Lettuce Big-Vein Virus

Lettuce big-vein disease, which is found in major lettuce production areas worldwide, is caused by Mirafiori lettuce big-vein virus (MLBVV). In order to produce marker-free transgenic lettuce resistant to MLBVV, we constructed a two T-DNA binary vector in which the first T-DNA contained a selectable marker gene npt II (neomycin phosphotransferase II) and the second T-DNA contained polyubiquitin gene promoter/terminator and inverted repeats of the viral coat protein (CP) gene. These T-DNAs were transferred via Agrobacterium tumefaciens (LBA4404)-mediated transformation into a lettuce cultivar ‘Watson’. About 40% of regenerated plants (T₀ generation) on kanamycin medium showed CP gene-positive by PCR analysis. CP gene-positive plants were self-pollinated, and 124 T₁ lines were analyzed for resistance to MLBVV. Twenty-one lines were selected as resistant to MLBVV, and 5 of 21 lines contained npt II-negative plants. Npt II-negative plants were self-pollinated, and 5 T₂ lines were analyzed for resistance to MLBVV. All lines showed resistance to MLBVV, but one line showed npt II-positive by PCR analysis (selection error in T₁ generation). The other 4 lines showed npt II-negative by both PCR and Southern blot analysis. Southern blot analysis showed that 2 of 4 lines contained one copy of the transgene per genome and that the other 2 lines contained more than one copy.