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Impact of wheat GRF4-GIF1 morphogenic regulators on transformation and genome editing efficiency in elite barley cultivars.

Ekaterina M Timonova·Antonina A Kiseleva·Mihail A Nesterov·Anna V Korotkova·Irina G Adonina·Alex V Kochetov·Elena A Salina
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Abstract

INTRODUCTION: Efficient genetic transformation is essential for the delivery of the CRISPR/Cas9 genome editing system and thus represents an important technology for breeding-oriented research in barley (Hordeum vulgare L.). However, transformation and plant regeneration from tissue culture remain challenging in non-model barley genotypes. Previous studies demonstrated that expression of a chimeric fusion between two interacting transcription factors, GROWTH-REGULATING FACTOR 4 (GRF4) and GRF-INTERACTING FACTOR 1 (GIF1), enhances regeneration capacity in wheat and other species. METHODS: In this study, we evaluated the effect of the wheat-derived GRF4-GIF1 morphogenic regulators on biolistic transformation and genome editing efficiency in three commercial barley cultivars: Tselinniy 5, Aley, and G-23035. RESULTS: The JD633 construct carrying GRF4-GIF1 enabled recovery of stable transformants in all three genotypes, with efficiencies ranging from 2.5% to 5%, whereas the control construct lacking morphogenic regulators resulted in no transgenic events in any of the tested varieties. Among transformed T0 plantlets, genome editing efficiency reached 64.3%, with predominantly biallelic mutations that were stably inherited in the T1 generation. Molecular screening revealed the presence of plasmid-free edited plants in the T0 generation, likely arising from transient Cas9 expression, and provided evidence of tissue chimerism. DISCUSSION: These results demonstrate that the GRF-GIF system facilitates genome editing, providing a practical framework for accelerating precision breeding in barley.

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CRISPR/Cas9GRF4-GIF1TransIT-2020biolisticsgenome editinggenotype independencemorphogenic regulatorsJournal Article

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Ekaterina M Timonova, Antonina A Kiseleva, Mihail A Nesterov, Anna V Korotkova, Irina G Adonina, Alex V Kochetov, Elena A Salina. 2026-08-31. Impact of wheat GRF4-GIF1 morphogenic regulators on transformation and genome editing efficiency in elite barley cultivars.. https://doi.org/10.3389/fpls.2026.1837794

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