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Summary  Development of marker‐free and transgene insertion site‐defined ( MFTID ) transgenic plants is essential for safe application of transgenic crops. However,  MFTID  plants have not been reported for wheat ( Triticum aestivum ). Here, we prepared a  RNA i cassette for suppressing lipoxygenase ( LOX ) gene expression in wheat grains using a double right border T‐ DNA  vector. The resultant construct was introduced into wheat genome via  Agrobacterium ‐mediated transformation, with four homozygous marker‐free transgenic lines (namely  GLRW ‐1, ‐3, ‐5 and ‐8) developed. Aided by the newly published wheat genome sequence, the T‐ DNA  insertion sites in  GLRW ‐3 and  GLRW ‐8 were elucidated at base‐pair resolution. While the T‐ DNA  in  GLRW ‐3 inserted in an intergenic region, that of  GLRW ‐8 inactivated an endogenous gene, which was thus excluded from further analysis. Compared to wild ‐type ( WT ) control,  GLRW ‐1, ‐3 and ‐5 showed decreased   LOX   gene expression, lower  LOX  activity and less lipid peroxidation in the grains; they also exhibited significantly higher germination rates and better seedling growth after artificial ageing treatment. Interestingly, the three  GLRW  lines also had substantially increased contents of several fatty acids (e.g., linoleic acid and linolenic acid) in their grain and flour samples than  WT  control. Collectively, our data suggest that suppression of grain  LOX  activity can be employed to improve the storability and fatty acid content of wheat seeds and that the  MFTID  line  GLRW ‐3 is likely of commercial value. Our approach may also be useful for developing the  MFTID  transgenic lines of other crops with enhanced grain storability and fatty acid content.

plant biotechnology journal

Development and characterization of marker‐free and transgene insertion site‐defined transgenic wheat with improved grain storability and fatty acid content