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The 2010 ASHS Annual Conference

3264:
Production of Marker-Free Transgenic Lettuce with Resistance to Mirafiori Lettuce Big-Vein Virus

Wednesday, August 4, 2010
Springs F & G
Yoichi Kawazu, National Institute of Vegetable and Tea Science, Mie 514-2392, Japan
Ryoi Fujiyama, National Institute of Vegetable and Tea Science, Mie 514-2392, Japan
Shunsuke Imanishi, National Institute of Vegetable and Tea Science, Mie 514-2392, Japan
Hirotaka Yamaguchi, National Institute of Vegetable and Tea Science, Mie 514-2392, Japan
Hiroyuki Fukuoka, National Institute of Vegetable and Tea Science, Mie 514-2392, Japan
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.
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