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Plant Biotechnol J. 2019 May 29. doi: 10.1111/pbi.13178. [Epub ahead of print]

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

Cao X1,2, Dong Z1, Tian D1,2, Dong L1, Qian W1, Liu J1, Liu X1, Qin H1, Zhai W1, Gao C1,2, Zhang K1,3, Wang D1,2,3.

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State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.
University of Chinese Academy of Sciences, Beijing, China.
College of Agronomy and State Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou, China.


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 RNAi 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.


RNA interference; common wheat; fatty acid content; lipoxygenase; marker-free transgenic plants; storability

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