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Recovery of transgenic plants by pollen-mediated transformation in Brassica juncea

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Abstract

The aroA-M1 encoding the mutant of 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) was introduced into the Brassica juncea genome by sonication-assisted, pollen-mediated transformation. The plasmid DNA and collected pollen grains were mixed in 0.3 mol/L sucrose solution and treated with mild ultrasonication. The treated pollen was then pollinated onto the oilseed stigmas after the stamens were removed artificially. Putative transgenic plants were obtained by screening germinating seeds on a medium containing glyphosate. Southern blot analysis of glyphosate-resistant plants indicated that the aroA-M1 gene had been integrated into the oilseed genome. Western blot analysis further confirmed that the EPSPS coded by aroA-M1 gene was expressed in transgenic plants. The transgenic plants exhibited increased resistance to glyphosate compared to untransformed plants. Some of those transgenic plants had considerably high resistance to glyphosate. The genetic analysis of T1 progeny further confirmed that the inheritance of the introduced genes followed the Mendelian rules. The results indicated that foreign genes can be transferred by pollen-mediated transformation combined with mild ultrasonication.

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Abbreviations

EPSPS:

5-Enolpyruvyl-shikimate-3-phosphate synthase

PCR:

Polymerase chain reaction

CTAB:

Cetyl trimethyl ammonium bromide

BSA:

Bovine serum albumin

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Acknowledgments

We deeply appreciate the provision of inbred oilseed lines and assistance with genetic transformation by Mr Genke Liu and Mr Xuejin Feng of the Cold Crops Institute at the Shanxi Academy of Agricultural Sciences, China. We also deeply appreciate the provision of the plasmid pGRA1300, and some help by Prof. Peilin Xu of Sun Yat-sen University, China.

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  1. School of Life Science and Technology, Shanxi University, Taiyuan, 030006, P.R. China

    Jingxue Wang, Yonghu Li & Chao Liang

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  1. Jingxue Wang
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  2. Yonghu Li
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  3. Chao Liang
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Correspondence to Jingxue Wang.

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Wang, J., Li, Y. & Liang, C. Recovery of transgenic plants by pollen-mediated transformation in Brassica juncea . Transgenic Res 17, 417–424 (2008). https://doi.org/10.1007/s11248-007-9115-x

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  • DOI: https://doi.org/10.1007/s11248-007-9115-x

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