Nitrogen (N) fertilizers have been widely applied to increase grain yield, as nitrogen is the major determinant of wheat crop yield and quality. However, excessive nitrogen fertilizers application aggravating environmental pollution and leads toward higher production cost. Therefore improving nitrogen use (NUE) is critical for sustainable wheat crop production.
BT1 a member of Bric-a-Brac/Tramtrack-Broad (BTB) gene family and negatively regulates nitrate transporters in model and crop plants. Under N limiting conditions Arabidopsis thaliana and rice (Oryza sativa) bt1 mutants exibited increased N uptake, as BT1 decrease the expression of NRT2.1 and NRT2.4 nitrate transporters. However, BT1 orthologs remained unidentified in wheat. In this study, four BT1 orthologs were identified in hexaploid wheat genome through plants.ensembl.org online BLASTN and MEGA7 alignment explorer tools. Single short guide RNAs (sgRNAs) were designed to knockout BT1 orthologs in wheat genome by CRISPR/Cas9 gene editing approach by focussing highly conserved regions.
The sgRNAs were expressed under U6 promoter and cloned in pJA32 final destiny plasmid vector. Agrobacterium tumefaciense hyper virulent strain EHA105 was transformed with sgRNAs gene construct and used for transformation of immature wheat embryos. 9-14 days old (post anthesis) immature wheat embryos demonstrated high callus formation and regeneration efficiency. Successful callus transformation and their conversion to tiny young plantlets were confirmed through PCR amplification, hygromycin selection assay and total N contents. Mutant wheat plants harboring missense or frameshift mutation in BT1 orthologous regions of wheat would reveal the reduction in N fertilizer requirement and potentially increase the grain yield in wheat crop.