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

Abstract

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. 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. 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 T 0 plantlets, genome editing efficiency reached 64.3%, with predominantly biallelic mutations that were stably inherited in the T 1 generation. Molecular screening revealed the presence of plasmid-free edited plants in the T 0 generation, likely arising from transient Cas9 expression, and provided evidence of tissue chimerism. These results demonstrate that the GRF-GIF system facilitates genome editing, providing a practical framework for accelerating precision breeding in barley.