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Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses

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Abstract

Plant defense responses can lead to altered metabolism and even cell death at the sites of Agrobacterium infection, and thus lower transformation frequencies. In this report, we demonstrate that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies in perennial ryegrass (Lolium perenne L.). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment and the addition of l-Gln prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies. Nearly 60 % of the stably transformed calluses regenerated into green plantlets. Calluses cultured on media lacking myo-inositol also displayed profound physiological and biochemical changes compared to ones cultured on standard growth media, such as reduced lignin within the cell walls, increased starch and inositol hexaphosphate accumulation, enhanced Agrobacterium binding to the cell surface, and less H2O2 production after Agrobacterium infection. Furthermore, the cold treatment greatly reduced callus browning after infection. The simple modifications described in this report may have broad application for improving genetic transformation of recalcitrant monocot species.

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Acknowledgments

We are grateful to Jin Tong for assistance in plant transformation, Dr. Minesh Patel for assistance with the Southern blots, Dr. Ann G. Matthysse of the University of North Carolina, Chapel Hill, for discussions on Agrobacterium binding, and Dr. Zhen-Ming Pei of Duke University for discussions on calcium signaling. We also thank Dr. Stan Gelvin for providing Agrobacterium strain A208. This work was supported by a grant from Bayer CropScience LP (Research Triangle Park, NC, USA).

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  1. Department of Grassland Science, China Agricultural University, Beijing, 100193, China

    Wan-Jun Zhang

  2. Crop Science Department, North Carolina State University, Campus Box 7287, Raleigh, NC, 27695, USA

    Wan-Jun Zhang, Ralph E. Dewey & Rongda Qu

  3. Plant Biology Department, North Carolina State University, Campus Box 7649, Raleigh, NC, 27695-7649, USA

    Wendy Boss & Brian Q. Phillippy

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  1. Wan-Jun Zhang
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Correspondence to Wan-Jun Zhang.

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Zhang, WJ., Dewey, R.E., Boss, W. et al. Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses. Plant Mol Biol 81, 273–286 (2013). https://doi.org/10.1007/s11103-012-9997-8

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