Abstract

Weed infestation is one of the crucial biotic stress factors contributing to yield loss in crop plants, more so in maize. Glyphosate {N-(phosphonomethyl)-glycine} is potent and most widely used broad- spectrum herbicide that interferes with shikimate pathway in the chloroplasts by inhibiting 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS). In plants and most bacterial EPSPS enzymes there exists a conserved motif crucial for binding phosphoenolpyruvate (PEP) or its competitive inhibitor glyphosate, the active ingredient in Roundup. Here, we report the developing of efficient herbicide resistant maize lines by CRISPR-Cas9 mediated site-specific EPSPS native gene fragment replacement via knock-out and knock-in with desired Homology Donor Repair (HDR) template. Using modified pCAMBIA vector backbone, we designed plasmid that harbours cassettes expressing plant codon optimized Cas9 from S. pyogenes and ZmEPSPS-sgRNAs. Homology donor plasmid was assembled in pZmU3 vector. The sgRNAs+pCas9 and HDR constructs were transformed into maize calli by using biolistic approach. Putative T 0 edited maize lines were validated by Cas9 and mEPSPS nested PCR analyses. T 1 seedlings of edited lines grew happily on media with glyphosate. Further, 30- d-old edited maize lines tolerated up to with 8ml/L of Roundup Ready. Endogenous shikimate levels and necrosis were quantified in these edited lines by HPLC that exhibited low shikimate levels (280- 2000 µmol/ml) in comparison to treated WT (6000 µmol/ml). Edited plants maintained enhanced photosynthetic capacity, transpiration rates, chlorophyll content and other related photosynthetic parameters like electron transport rate, maximal photosystem II efficiency (Fv/Fm) in comparison to treated-WT. Results revealed enhanced glyphosate resistance in our edited maize lines with no yield penalty.