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Transgene integration complexity and expression stability following biolistic or Agrobacterium-mediated transformation of sugarcane

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Abstract

Sugarcane (Saccharum spp. hybrids) accounts for 80% of the table sugar produced worldwide and is also a prime feedstock for biofuel production. However, very few studies are available for directly comparing Agrobacterium tumefaciens-mediated transfer of T-DNA (AMT) and biolistic transfer of minimal expression cassettes (BLT MC) regarding transgene complexity and expression stability. In this study, the transformation efficiency, transgene integration pattern, expression level, and expression stability were compared in the commercially important sugarcane cultivar CP88-1762. A total of 312 transgenic lines derived from AMT and 250 lines derived from BLT MC were identified by PCR from genomic DNA using nptII-specific primers. Lines were analyzed with both qPCR (TaqMan®) and NPTII ELISA to determine the nptII transgene copy number and expression level. The results of Southern blot analysis on selected lines were highly correlated to the qPCR results. There were no significant differences between the two transformation systems for transformation efficiency, frequency of single copy integration, or level and stability of transgene expression when carried out with the same expression cassette, tissue culture, and selection procedure in 12 independent experiments. These findings suggested that both BLT MC and AMT provide suitable platforms for generation of elite sugarcane events.

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Acknowledgments

The authors would like to thank Syngenta for financial support, Dr. Robert Gilbert (Everglades Research and Educational Center, UF-IFAS, Belle Glade, FL) for providing tops of sugarcane cultivar CP88-1762 and Sun Gro Horticulture, Apopka, FL for donation of the Fafard # 2 potting mix.

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Authors and Affiliations

  1. Agronomy Department, Plant Molecular and Cellular Biology Program, University of Florida, IFAS, PO Box 110300, Gainesville, Florida, 32611, USA

    Hao Wu, Faisal Saeed Awan, Aloisio Vilarinho, Qianchun Zeng, Baskaran Kannan, Tenisha Phipps & Fredy Altpeter

  2. Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, 38000, Pakistan

    Faisal Saeed Awan

  3. Embrapa Roraima, P.O.Box 133, Boa Vista, RR, 69301-970, Brazil

    Aloisio Vilarinho

  4. Biochemistry and Biotechnology Department, Yunnan Agricultural University, Kunming, 650201, China

    Qianchun Zeng

  5. Syngenta, Research Triangle Park, Durham, NC, 27709, USA

    Jamie McCuiston, Wenling Wang & Kerry Caffall

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  1. Hao Wu
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  2. Faisal Saeed Awan
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  10. Fredy Altpeter
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Correspondence to Fredy Altpeter.

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Editor: John Finer

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Wu, H., Awan, F.S., Vilarinho, A. et al. Transgene integration complexity and expression stability following biolistic or Agrobacterium-mediated transformation of sugarcane. In Vitro Cell.Dev.Biol.-Plant 51, 603–611 (2015). https://doi.org/10.1007/s11627-015-9710-0

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