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Inside out: high-efficiency plant regeneration and Agrobacterium-mediated transformation of upland and lowland switchgrass cultivars

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Abstract

Key message

Selection of pre-embryogenic callus from a core structure from mature seed-derived callus is the key for high-efficiency plant regeneration and transformation of switchgrass different cultivars.

Abstract

Switchgrass (Panicum virgatum L.) has been identified as a dedicated biofuel crop. For its trait improvement through biotechnological approaches, we have developed a highly efficient plant regeneration and genetic transformation protocol for both lowland and upland cultivars. We identified and separated a pre-embryogenic “core” structure from the seed-derived callus, which often leads to development of highly regenerative type II calluses. From the type II callus, plant regeneration rate of lowland cultivars Alamo and Performer reaches 95 %, and upland cultivars Blackwell and Dacotah, 50 and 76 %, respectively. The type II callus was also amenable for Agrobacterium-mediated transformation. Transformation efficiency of 72.8 % was achieved for lowland cultivar Alamo, and 8.0 % for upland cultivar Dacotah. PCR, Southern blot and GUS staining assays were performed to verify the transgenic events. High regenerative callus lines could be established in 3 months, and transgenic plants could be obtained in 2 months after Agrobacterium infection. To our knowledge, this is the first report on successful plant regeneration and recovery of transgenic plants from upland switchgrass cultivars by Agrobacterium-mediated transformation. The method presented here could be helpful in breaking through the bottleneck of regeneration and transformation of lowland and upland switchgrass cultivars and probably other recalcitrant grass crops.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

BAP:

6-Benzylaminopurine

CH:

Casein hydrolysate

GA:

Gibberellin

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Acknowledgements

The work was supported by the National High Technology Research and Development Program (“863” 2012AA101801) of China, and Chinese Universities Scientific Funds (2012QJ120) to W.J.Z. We are grateful to Dr. Rongda Qu of NCSU for his critical reading and advices on the paper.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Grassland Science, China Agricultural University, No. 2 Yuanmingyuan Xilu, Haidian District, Beijing, 100193, People’s Republic of China

    Yan-Rong Liu, Hui-Fang Cen, Jian-Ping Yan, Yun-Wei Zhang & Wan-Jun Zhang

  2. National Energy R&D Center for Biomass (NECB), China Agricultural University, No. 2 Yuanmingyuan Xilu, Haidian District, Beijing, 100193, People’s Republic of China

    Yun-Wei Zhang & Wan-Jun Zhang

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  1. Yan-Rong Liu
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  2. Hui-Fang Cen
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  4. Yun-Wei Zhang
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Correspondence to Wan-Jun Zhang.

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Communicated by K. Wang.

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Liu, YR., Cen, HF., Yan, JP. et al. Inside out: high-efficiency plant regeneration and Agrobacterium-mediated transformation of upland and lowland switchgrass cultivars. Plant Cell Rep 34, 1099–1108 (2015). https://doi.org/10.1007/s00299-015-1769-x

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  • DOI: https://doi.org/10.1007/s00299-015-1769-x

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