Figure 3
Progressive loss of transgene expression in subsequent generations. A, Gene expression analysis in homozygous plants of 5 independent N. attenuata lines, comparing two consecutive generations (T2 and T3). Transcript abundance of the ICE and PNA transgenes was determined by qRT-PCR on cDNA of rosette-stage leaves (30 dpg). Bars indicate the ΔCT mean expression (log2 fold expression) relative to actin as the reference gene (±SD, n = 3 plants). The T3 generation of the lines ICE 4.4.1, PNA 1.2.1 and PNA 10.1.1 indicated a strong reduction of transgene expression. Lines ICE 1.1.1 and PNA 8.6.1 showed stable and high transgene expression in the T3 generation. B, Photographs of 15-day-old T3 seedlings grown on a hygromycin B containing GB5 medium. Sensitivity to hygromycin was indicated by small, yellowish seedlings and the lack of root hairs. Resistant seedlings were larger, with dark green cotyledons and clearly developed root hairs. The lines ICE 4.4.1, PNA 1.2.1 and PNA 10.1.1 showed sensitivity to hygromycin in the T3 generation similar to wild-type, whereas the lines ICE 1.1.1 and PNA 8.6.1 remained hygromycin resistance. For each line seeds of three different plants were analyzed and wild-type seeds used as negative control.