Figure 8

Recovery of transgene expression after secondary regeneration. A, Secondary callus regeneration was performed from hypocotyls of homozygous T₂ seedlings from the lines PNA 1.2, ICE 4.4 and ir-ACX1. From the 54 secondary regenerated plants 22 produced offspring which showed full resistance to hygromycin. In the T₄ stage the offspring from 9 lines remained resistant. B, Gene expression analysis after secondary regeneration. Fully resistant offspring were grown in the glasshouse and qRT-PCR was performed on leaf cDNA from rosette-stage plants. For each genotype conventionally propagated T₃ plants were included as positive and negative controls, respectively. Bars indicate the ΔC_T mean expression (log₂ fold expression) of the ectopically expressed PNA and ICE transgenes relative to actin as the reference gene (±SD, n = 3 plants). C, Hygromycin sensitivity of T₄ seedlings (direct descendants of the plants used for gene expression) indicates an ongoing silencing process (±SD, n = 3 plants).