Stable transgene expression and random gene silencing in wheat

Abstract

Wheat genes for pathogenesis-related (PR-)proteins, chitinase and beta-1,3-glucanase, under the control of maize ubiquitin promoter-intron were used for transforming the spring wheat 'Bobwhite', using a biolistic approach. Twenty of the 24 primary transgenic lines expressing the PR-protein genes in the T0 generation were silenced in either the T1 or T2 generations. Two apparently genetically identical regenerants arising from a single callus co-bombarded with chitinase and beta-1,3-glucanase transgene combinations, but differing in the expression of the transgenes were selected for further characterization. In one homozygous line, transgene silencing was observed in the T3 plants, while the other line homozygous for the transgene loci stably expressed and inherited the transgenes to at least the T4 generation. Southern blot analyses of genomic DNA from the two lines using the isoschizomeric methylation-sensitive enzymes, MspI and HpaII, revealed a higher degree of methylation of CCGG sequences in the line with the silenced transgene locus. Analysis by reverse transcriptase-polymerase chain reaction, Northern blotting and Western blotting detected stable expression of the transgenes in the line with a lesser extent of methylation, whereas the line with a higher level of CCGG methylation had no transgene expression by the T3 generation. The germination of seeds from the silenced plants in the presence of a cytidine analogue, 5-azacytidine (azaC), did not lead to a reversion of this phenotype.