Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Aug;119(4):2116-2132.
doi: 10.1111/tpj.16880. Epub 2024 Jun 23.

Use of GRF-GIF chimeras and a ternary vector system to improve maize (Zea mays L.) transformation frequency

Wout Vandeputte  1   2 , Griet Coussens  1   2 , Stijn Aesaert  1   2 , Jari Haeghebaert  1   2 , Lennert Impens  1   2 , Mansour Karimi  1   2 , Juan M Debernardi  3 , Laurens Pauwels  1   2
Affiliations
Free article

Use of GRF-GIF chimeras and a ternary vector system to improve maize (Zea mays L.) transformation frequency

Wout Vandeputte et al. Plant J. 2024 Aug.
Free article

Abstract

Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium-mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene-editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1-VIR2 virulence helper plasmid to improve T-DNA delivery, and/or expressing a fusion protein between a GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1-VIR2 helper vector with no effect on event quality regarding T-DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5- to 6.5-fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR-/Cas9-mediated gene editing. Our results demonstrate how a GRF-GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene-editing applications and molecular biology studies in maize.

Keywords: B104; CRISPR/Cas; GRF‐GIF; Zea mays; gene editing; hygromycin B; morphogenic genes; technical advance; ternary vector system; transformation.

PubMed Disclaimer

References

REFERENCES

    1. Aesaert, S., Impens, L., Coussens, G., Van Lerberge, E., Vanderhaeghen, R., Desmet, L. et al. (2022) Optimized transformation and gene editing of the B104 public maize inbred by improved tissue culture and use of morphogenic regulators. Frontiers in Plant Science, 13, 883847. Available from: https://doi.org/10.3389/fpls.2022.883847
    1. Anand, A., Bass, S.H., Wu, E., Wang, N., McBride, K.E., Annaluru, N. et al. (2018) An improved ternary vector system for Agrobacterium‐mediated rapid maize transformation. Plant Molecular Biology, 97(1–2), 187–200. Available from: https://doi.org/10.1007/s11103‐018‐0732‐y
    1. Andorf, C., Beavis, W.D., Hufford, M., Smith, S., Suza, W.P., Wang, K. et al. (2019) Technological advances in maize breeding: past, present and future. Theoretical and Applied Genetics, 132(3), 817–849. Available from: https://doi.org/10.1007/s00122‐019‐03306‐3
    1. Barone, P., Wu, E., Lenderts, B., Anand, A., Gordon‐Kamm, W., Svitashev, S. et al. (2020) Efficient gene targeting in maize using inducible CRISPR‐Cas9 and marker‐free donor template. Molecular Plant, 13(8), 1219–1227. Available from: https://doi.org/10.1016/j.molp.2020.06.008
    1. Beringer, J., Chen, W., Garton, R., Sardesai, N., Wang, P.‐H., Zhou, N. et al. (2017) Comparison of the impact of viral and plant‐derived promoters regulating selectable marker gene on maize transformation and transgene expression. Plant Cell Reports, 36(4), 519–528. Available from: https://doi.org/10.1007/s00299‐017‐2099‐y

MeSH terms

LinkOut - more resources