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Development and validation of a novel and robust cell culture system in soybean (Glycine max (L.) Merr.) for promoter screening

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A novel soybean cell culture was developed, establishing a reliable and rapid promoter assay to enable high-throughput automated screening in soybean protoplasts relevant to shoot tissues in whole plants.

Abstract

Transient reporter gene assays can be valuable to rapidly estimate expression characteristics of heterologous promoters. The challenge for maximizing the value of such screens is to combine relevant cells or tissues with methods that can be scaled for high-throughput screening, especially for crop—rather than model species. We developed a robust and novel soybean cell suspension culture derived from leaf-derived callus for protoplast production: a platform for promoter screening. The protoplasts were transfected with promoter–reporter constructs, of which were chosen and validated against known promoter expression profiles from tissue-derived protoplasts (leaves, stems, and immature cotyledons) and gene expression data from plants. The cell culture reliably produced 2.82 ± 0.94 × 108 protoplasts/g fresh culture mass with a transfection efficiency of 31.06 ± 7.69% at 48 h post-incubation. The promoter–reporter gene DNA expression levels of transfected cell culture-derived protoplasts were most similar to that of leaf- and stem-derived protoplasts (correlation coefficient of 0.99 and 0.96, respectively) harboring the same constructs. Cell culture expression was also significantly correlated to endogenous promoter-gene expression in leaf tissues as measured by qRT-PCR (correlation coefficient of 0.80). Using the manual protocols that produced these results, we performed early stage experiments to automate protoplast transformation on a robotic system. After optimizing the protocol, we achieved up to 29% transformation efficiency using our robotic system. We conclude that the soybean cell culture-to-protoplast transformation screen is amenable to automate promoter and gene screens in soybean that could be used to accelerate discoveries relevant for crop improvement. Key features of the system include low-cost, facile protoplast isolation, and transformation for soybean shoot tissue-relevant molecular screening.

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Acknowledgements

The authors wish to thank Dr. Wusheng Liu for providing soybean seeds and Dr. Agnieszka A. Piatek for help in plasmid isolation and cloning. The authors also wish to thank Jake Massengill for early stage assistance with robotics and Mary-Anne Nguyen for later assistance. This study was supported by funding from the Tennessee Soybean Promotion Board. We also appreciate support from a USDA Hatch Grant.

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Author notes

  1. Taylor P. Frazier

    Present address: Elo Life Systems, Suite Number 2200, 3054 E Cornwallis Road, Durham, NC, 27709, USA

Authors and Affiliations

  1. Department of Plant Sciences, University of Tennessee, Knoxville, TN, USA

    Mst. Shamira Sultana, Taylor P. Frazier, Reginald J. Millwood & C. Neal Stewart Jr.

  2. Center for Agricultural Synthetic Biology, University of Tennessee Institute of Agriculture, Knoxville, TN, USA

    Mst. Shamira Sultana, Taylor P. Frazier, Scott C. Lenaghan & C. Neal Stewart Jr.

  3. Department of Food Science, University of Tennessee, Knoxville, TN, USA

    Scott C. Lenaghan

  4. Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville, TN, USA

    Scott C. Lenaghan

Authors
  1. Mst. Shamira Sultana
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  4. Scott C. Lenaghan
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  5. C. Neal Stewart Jr.
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Contributions

MSS participated in the design, established cell cultures, conducted all experiments, analyzed data, and drafted the manuscript. TPF participated in the design, helped to optimize protoplast transformation experiments, and assisted to troubleshoot experiments, data analysis, and manuscript revisions. RJM assisted with the valuable suggestions for the experiments, data analysis, and manuscript revision. SCL was responsible for valuable discussion of designing the plasmid, revising, and writing the manuscript. CNS conceived and coordinated the study, responsible for revising and writing the manuscript, data analysis, and obtained the funding. All authors have read and agree with the content of manuscript.

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Correspondence to C. Neal Stewart Jr..

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Conflict of interest

The authors are inventors on a relevant U.S. patent application that has been assigned to the University of Tennessee Research Foundation.

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Communicated by Baochun Li.

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Sultana, M.S., Frazier, T.P., Millwood, R.J. et al. Development and validation of a novel and robust cell culture system in soybean (Glycine max (L.) Merr.) for promoter screening. Plant Cell Rep 38, 1329–1345 (2019). https://doi.org/10.1007/s00299-019-02455-5

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