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Long-term T-DNA insert stability and transgene expression consistency in field propagated sugarcane

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Abstract

Key message

This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. T-DNA inserts are stable; no transgene rearrangements were observed. AmCYAN1 and PMI protein accumulation levels were maintained. There was no evidence that production of either protein declined across generations and no transgene silencing was observed in three commercial sugarcane varieties through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes over 4 years of field testing. Long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized.

Abstract

This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. These data are critical supporting information needed for successful commercialization of GM sugarcane. Here seventeen transgenic events, containing the AmCYAN1 gene driven by a CMP promoter and the E. coli PMI gene driven by either a CMP or Ubi promoter, were used to monitor T-DNA insert stability and consistency of transgene encoded protein accumulation through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes. The experiments were conducted in three commercial sugarcane varieties over 4 years of field testing. DNA gel blot analysis showed that the T-DNA inserts are stable; no transgene rearrangements were observed. Quantitative ELISA showed no evidence of decreasing AmCYAN1 and PMI protein levels across generations and no transgene silencing was observed. These results indicate that long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized.

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Abbreviations

GM:

Genetically modified

AmCYAN1:

Anemonia majano cyan fluorescent protein

PMI:

E. coli phosphomannose isomerase

CMP:

Cestrum yellow leaf curling virus promoter

Ubi:

Maize polyubiquitin1 promoter

ELISA:

Enzyme-linked immuno-sorbent assay

tNOS:

Terminator of nopaline synthase gene

T0:

Plants generated from transformation

T-DNA:

Transfer DNA

CTAB:

Cetyl trimethylammonium bromide

DIG:

Digoxigenin

NPTII:

Neomycin phosphotransferase

PAT:

Phosphinothricin acetyl-transferase

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Acknowledgements

We thank Michael Nuccio, David Patton, Tichafa Munyikwa, Emily Smith (produced the graphs 4–9), Doug Russell, Lauren De Carbonnel, Sivamani Elumalai, Anic Deframond and Kasi Azhakanandam for edits, helpful suggestions and comments.

Author contributions

Kerry Hosmer Caffall designed the experiments, did sampling, analyzed the data and drafted the manuscript. Chengkun He coordinated the multiple function groups, performed data analysis, wrote and finalized the manuscript. Michele Smith-Jones designed field experiments, did sampling and edited the manuscript. Kris Mayo, Pearl Mai, Shujie Dong, John Ke, and Erik Dunder conducted sugarcane transformation and edited the manuscript. Michele Yarnall and Rachel Whinna generated protein ELISA data and edited the manuscript. Joe DeMaio and Weining Gu designed Southern blotting experiments, generated the Southern blotting data and edited the manuscript. Judith Sheldon generated the AmCYAN1 protein expression data by confocal microscopy analyses. Martin Allen, Tricia Costello, and Ada Snyder did sampling and assay submission. Kristin Setliff, Rakesh Jain, Clark Lovelady, Erik Rawls, and Erik Palmer provided sugar cane field testing. Yan Zhang, Nicholas Bate, Liang Shi, and Ian Jepson provided the project support and edited the manuscript.

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Authors and Affiliations

  1. Syngenta Crop Protection, LLC, 9 Davis Drive, Research Triangle Park, Durham, NC, 27709-2257, USA

    Kerry Hosmer Caffall, Chengkun He, Kristin Mayo, Pearl Mai, Shujie Dong, John Ke, Erik Dunder, Michele Yarnall, Rachel Whinna, Joe DeMaio, Weining Gu, Martin Allen, Tricia Costello, Kristin Setliff, Yan Zhang, Nicholas Bate, Liang Shi & Ian Jepson

  2. Syngenta Seeds Incorporated, 2369 330th Street, Slater, IA, 50244, USA

    Michele Smith-Jones

  3. Vero Beach Research Center, Syngenta Crop Protection, LLC, 7145 58th Avenue, Vero Beach, FL, 32967, USA

    Rakesh Jain, Ada Snyder, Clark Lovelady, Eric Rawls & Eric Palmer

  4. Syngenta Jealott’s Hill Research Center, Bracknell, BRK, RG42 6EY, UK

    Judith Sheldon

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  1. Kerry Hosmer Caffall
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  14. Martin Allen
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  22. Yan Zhang
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Corresponding author

Correspondence to Chengkun He.

Additional information

Kerry Hosmer Caffall and Chengkun He are the co-first authors.

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Caffall, K.H., He, C., Smith-Jones, M. et al. Long-term T-DNA insert stability and transgene expression consistency in field propagated sugarcane. Plant Mol Biol 93, 451–463 (2017). https://doi.org/10.1007/s11103-016-0572-6

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  • DOI: https://doi.org/10.1007/s11103-016-0572-6

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