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Comparison of transformation frequency using the bar gene regulated by the CaMV 35S or NOS promoter in Agrobacterium-mediated soybean (Glycine max L.) transformation

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Abstract

In Agrobacterium tumefaciens-mediated plant transformation, the promoter chosen to drive a selectable marker gene has an effect on transformation frequency. The objective of this work was to compare the effect on soybean transformation of different promoter and regulator combinations driving a selectable marker gene using mature seeds as explants. Two commonly used promoters, double cauliflower mosaic virus 35S (CaMV 35S) and nopaline synthase (NOS), and one regulator, tobacco etch virus (TEV) translational enhancer, were tested. Four different promoter/enhancer combinations were constructed to drive a bialaphos resistance gene (bar) and used for multiple independent transformation experiments. Herbicide application and PCR analysis were used to confirm the inheritance of the bar gene in the T 1 generation. Quantitative real-time PCR (qRT-PCR) was used to estimate transgene copy number in T 1 transgenic events. The average transformation frequency from combining NOS with the TEV enhancer (3.5% across 12 replications) was significantly higher than the frequencies obtained from the double CaMV 35S without an enhancer (1.6% across 16 replications), double CaMV 35S with an enhancer (1.4% across 8 replications), and NOS without an enhancer (1.0% across 12 replications). The bar transcript levels in T 1 transgenic leaf tissue did not correlate with transformation frequencies achieved by the different constructs. No significant differences were identified between constructs in the average transgene copy number across events. These data show that a selectable marker system comprised of the bar gene regulated by the NOS promoter in combination with the TEV enhancer is preferred in Agrobacterium-mediated soybean transformation.

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Acknowledgments

The authors thank Chichun Yang and Qing Ji for their assistance in constructing pTF101.2, pTF101.3, and pTF101.4. Graduate research (by A.T.) was supported by the US Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) grant (proposal number 2012-03598 to K.W.). Additional support was provided by the USDA-NIFA Hatch Project (No. IOW05162) and by State of Iowa funds (K.W., K.L., D.L.).

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  1. Interdepartmental Genetics and Genomics Major, Iowa State University, Ames, IA, 50011, USA

    Amber Testroet

  2. Department of Agronomy, Iowa State University, Ames, IA, 50011, USA

    Amber Testroet, Keunsub Lee, Diane Luth & Kan Wang

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  1. Amber Testroet
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Correspondence to Kan Wang.

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Editor: David R. Duncan

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Figure S1

Comparison of T-DNA copy number estimation in T 1 generation by qPCR and ddPCR. The T-DNA copy numbers determined by ddPCR were plotted against those estimated by SYBR-qPCR. A total of 10 plants with one to 31 copies of T-DNA were included in the analysis. A strong linear correlation was found: slope was 0.85 and the correlation coefficient (r) was 0.95. (PPTX 37 kb)

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Testroet, A., Lee, K., Luth, D. et al. Comparison of transformation frequency using the bar gene regulated by the CaMV 35S or NOS promoter in Agrobacterium-mediated soybean (Glycine max L.) transformation. In Vitro Cell.Dev.Biol.-Plant 53, 188–199 (2017). https://doi.org/10.1007/s11627-017-9810-0

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