CRISPR/Cas9 mediated TaRPK1 root architecture gene mutagenesis confers enhanced wheat yield

CRISPR/Cas9 system has emerged as an efficient tool for sustainable crop improvement. Roots are the "principal hidden organ" that has a crucial function in vascular plants. Receptor-like protein kinase 1 (RPK1) has been reported to regulate root architecture system (RAS), abiotic stress, and yield both in Arabidopsis and rice. We employed a CRISPR/Cas9-based system, namely LR-1 and LR-2 constructs having double guided RNAs transformed via agrobacterium for targeted mutagenesis of TaRPK1 genes to alter the root architecture and hence yield in Triticum aestivum. Sequencing confirmed seven CRISPR/Cas9-based mutated T0 lines of LR-1 constructs and six T0 lines of LR-2 constructs, with an overall mutation efficiency of 41.93%. The T0 plants displayed higher monoallelic mutation compared to the diallelic mutation. 37.5% monoallelic mutation at target site 1 within the D genome by gRNA1 was observed by the LR-1 construct. The LR-2 constructs showed a higher monoallelic mutation frequency of 26.67% at target sites 1 and 2 within A, B, and D genomes. The deletions were mainly short, however longer deletions such as 12d, 17d, 19d, and 20d were detected by gRNA2 of LR-1 construct. Transgenic lines revealed significant alteration in morphology and RSA with a significant increase in number of effective tillers, grain weight, root length, root depth, root volume, and root surface area while reduced root diameter, root angle, and spike length, compared to the wild plants. Tillers and total grain weight increased significantly, suggesting edited lines increased grain production by decreasing spike length. The study validates that CRISPR/Cas9 mediated targeted editing of TaRPK1 is a practical approach for modifying RAS and hence yield enhancement in wheat.