Gene editing of the representative WRKY family members in an elite malting barley cultivar RGT Planet by CRISPR/Cas9

Naghmeh Nejat

Barley is one of the world’s most economically important cereal crops and a superb model plant for genetic studies and elucidation of plant responses to environmental changes and stress-associated events due to its exceptional adaptations to broad climatic and environmental conditions. CRISPR/Cas system has a great potential for crop genetic improvement and has revolutionised genetics research and crop breeding. CRISPR-based gene targeting can accelerate the improvement of market-oriented traits and elite varieties through precision crop breeding. WRKY transcription factors (TFs), as a superfamily of TFs, constitute one of the largest transcription factor families in plants. WRKY TFs with diverse biological functions play a pivotal role in activating several developmental and physiological processes and fine-tuning signalling pathways and defence responses. However, little is known about the exact growth and developmental functions of WRKY family members in barley. In this study, 14 representative WRKY transcription factors were selected from different WRKY groups based on phylogenetic analysis. Pooled gRNAs were synthesised for multiplex CRISPR/Cas9 mutagenesis. gRNA constructs carrying one, two or three gRNA cassettes were assembled and delivered into RGT Planet, a high yielding spring malting barley with superior agronomic traits. A collection of 105 mutant lines with remarkable phenotypic variations were obtained. An average mutation frequency in the T0 population created by the pooled CRISPR/Cas9 was 89.74% containing multiple mutations. This study, for the first time, provided strong evidence of the roles of HvWRKY39 in tiller development and HvWRKY32 in spike development, grain filling, and kernel weight. Evaluation of yield components revealed the contribution of HvWRKY32 and HvWRKY39 toward increasing barley yield potential by approximately 20%. Our findings also demonstrated the roles of HvWRKY TFs in root development and root architecture. These results indicated that TFs have tremendous potential to enhance crop improvement and boost crop yield. This study established a promising genomic editing platform with the modern barley variety RGT Planet. The mutant library generated from this study provides a valuable resource for further detailed functional genomics in barley.

Gene editing of the representative WRKY family members in an elite malting barley cultivar RGT Planet by CRISPR/Cas9

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