RECURRENT ONSET OF EPIGENETIC SILENCING IN RICE HARBORING A MULTI-COPY TRANSGENE

Extensive methylation of the Ubi1 promoter has been shown to be associ ated with transcriptional silencing and bialaphos herbicide sensitivit y in several R-1 progeny derived from a transgenic rice line, JKA 52, containing multiple copies of the introduced genes (Kumpatla at al., 1 997; Plant Physiol. 115, 361-373). All of the progeny from silenced R- 1 lines were silenced, and a proportion (24%) of the R-2 seedlings fro m JKA 52-6, a non-silenced homozygous R-1 line, were found to be silen ced. Several non-silenced (bialaphos-resistant) R-2 progeny derived fr om the selfed seed of JKA 52-6 were tested at various stages throughou t the growth period and found to be resistant to bialaphos, indicating the absence of silencing. Five of these were investigated further to determine whether silencing could recur in subsequent generations. R-3 seedlings were raised from selfed R-2 Seed and herbicide sensitivity was detected in 5-40% of the plants in three of the five lines. This s ilencing was accompanied by methylation of the Ubi1 promoter and the a bsence of steady-state bar mRNA. This recurrent onset of silencing and instability of expression associated with a transgene locus containin g several repeated sequences is undesirable. The data strongly suggest that multi-generation molecular analysis should be undertaken for tra nsgenic lines to be used for agricultural purposes and that lines cont aining methylated inserts should be carefully evaluated before being i ncluded in further breeding strategies.