The production of male-sterile wheat plants through split barnase expression is promoted by the insertion of introns and flexible peptide linkers

The successful use of transgenic plants depends on the strong and stable expression of the heterologous genes. In this study, three introns (PSK7-i1 and PSK7-i3 from Petunia and UBQ10-i1 from Arabidopsis) were tested for their ability to enhance the tapetum-specific expression of a split barnase transgene. We also analyzed the effects of introducing multiple copies of flexible peptide linkers that bridged the fusion domains of the assembled protein. The barnase fragments were assembled into a functional cytotoxin via intein-mediated trans-splicing, thus leading to male sterility through pollen ablation. A total of 14 constructs carrying different combinations of introns and peptide linkers were transformed into wheat plants. The resulting populations (between 41 and 301 independent plants for each construct) were assayed for trait formation. Depending on which construct was used, there was an increase of up to fivefold in the proportion of plants exhibiting male sterility compared to the populations harboring unmodified constructs. Furthermore, the average barnase copy number in the plants displaying male sterility could be reduced. The metabolic profiles of male-sterile transgenic plants and non-transgenic plants were compared using gas chromatography–mass spectrometry. The profiles generated from leaf tissues displayed no differences, thus corroborating the anther specificity of barnase expression. The technical advances achieved in this study may be a valuable contribution for future improvement of transgenic crop systems.