Abstract
Genetic manipulation using gene cassettes was applied to the elite wheat variety EM12 via particle bombardment, which allows an improvement in transformation frequency. We simultaneously transferred to wheat immature embryos with two non-linked genes, gus and bar, on either separate gene cassettes or one plasmid. The linear gene cassettes were excised and purified by restriction digestion of the plasmid, and consisted of promoters, open reading frames and terminators. No difference was observed in GUS transient expression of between gene cassettes and single whole plasmid. However, the stable transformation frequency was significantly increased to 1.1% using gene cassettes, compared with 0.4% when using single plasmid. Procedures of the efficient co-transformation with gene cassettes were developed. Factors influencing on the transformation frequency were also studied in order to optimize the procedure. These were acceleration pressure, target distance, gold particle size, the quantity ratio of gene cassettes and the age of target explants. Based on the transient and stable expression of the gus gene cassettes, optimization of transformation parameters improved the reproducibility of transformation in the elite wheat variety.
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Acknowledgements
The work described here was initiated under the national basic research programme of China 2002 CB111302 “Functional genomics and molecular improvement of quality related traits in important agricultural crops” and the national natural science foundation of china 30370807 “cloning of tissue specific promoter in Tritordeum”. The authors thank greatly for Prof. Peter Shewry and John Cram giving suggestion and critical annotation.
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Yao, Q., Cong, L., He, G. et al. Optimization of wheat co-transformation procedure with gene cassettes resulted in an improvement in transformation frequency. Mol Biol Rep 34, 61–67 (2007). https://doi.org/10.1007/s11033-006-9016-8
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DOI: https://doi.org/10.1007/s11033-006-9016-8