The trans-silencing of L91 and E82 by C73 is stably inherited for up to five generations. Trans-silencing suppresses the cop1-like PTGS phenotype of L91 homozygotes and of E82 plants and causes reactivation of E82 GFP-COP1 expression. (A) Legend to illustrate the phenotypic scoring scheme. (B) Diagnostic PCR assay. DNA from individual F2 plants was subjected to PCR with primers specific for L91 and C73, or E82 and C73, as indicated. Lane 1: L91 or E82 control. Lane 10: C73 control. Lanes 2 to 9: Eight individual F2 plants. The gene dosage for L91 and E82 was defined by the segregation ratio of the kanamycin resistance marker in the F3 progeny (KanR:S, either 3:1, 15:1, or all). (C, D) Transgene expression (bars) and endogene silencing phenotypes (cop, wt) of parental controls, dihybrid F1 plants or their single-transgene control siblings, individually genotyped F2s, as well as their selfed progeny. (C) E82 and C73 and (D) L91 and C73. Shading of the bars reflects the percentage of plants with active or silenced GFP-COP1 transgene expression. In the case of heterozygous lineages (s) the average family size was 32. Note: In the F4 generation a small fraction of plants with a cop1-like cosuppression phenotype was seen among the majority of wild-type plants.