Transgene‐independent heredity of Rd DM ‐mediated transcriptional gene silencing of endogenous genes in rice

To induce transcriptional gene silencing ( TGS ) of endogenous genes of rice ( Oryza sativa L.), we expressed double‐strand RNA of each promoter region and thus induced RNA ‐directed DNA methylation (Rd DM ). We targeted constitutively expressed genes encoding calnexin ( CNX ), protein disulphide isomerase ( PDIL 1‐1 ) and luminal binding protein ( BiP1 ); an endoplasmic reticulum stress‐inducible gene ( Osb ZIP 50 ); and genes with seed‐specific expression encoding α‐globulin ( Glb ‐ 1 ) and glutelin‐B4 ( GluB4 ). TGS of four genes was obtained with high efficiency ( CNX , 66.7% of regenerated plants; OsBiP1 , 67.4%; Osb ZIP 50 , 63.4%; GluB4 , 66.1%), whereas the efficiency was lower for PDIL 1‐1 (33.3%) and Glb‐1 TGS lines (10.5%). The heredity of TGS , methylation levels of promoter regions and specificity of silencing of the target gene were investigated in some of the TGS lines. In progeny of CNX and Osb ZIP 50 TGS lines, suppression of the target genes was preserved (except in the endosperm) even after the removal of trigger genes (T‐ DNA ) by segregation. TGS of CNX was reverted by demethylation treatment, and a significant difference in CG and CHG methylation levels in the −1 to −250 bp region of the CNX promoter was detected between the TGS and revertant lines, suggesting that TGS is closely related to the methylation levels of promoter. TGS exhibited specific suppression towards the target gene compared with post‐transcriptional gene silencing when GluB4 gene from glutelin multigene family was targeted. Based on these results, future perspectives and problems to be solved in the application of Rd DM to new plant breeding techniques in rice are discussed.