Abstract
Weeds and their devastating effects have been a great threat since the start of agriculture. They compete with crop plants in the field and negatively influence the crop yield quality and quantity along with survival of the plants. Glyphosate is an important broad-spectrum systemic herbicide which has been widely used to combat various weed problems since last two decades. It is very effective even at low concentrations, and possesses low environmental toxicity and soil residual activity. However, the residual concentration of glyphosate inside the plant has been of major concern as it severely affects the important metabolic pathways, and results in poor plant growth and grain yield. In this study, we compared the glyphosate tolerance efficiency of two different transgenic groups over expressing proline/173/serine (P173S) rice EPSPS glyphosate tolerant mutant gene (OsmEPSPS) alone and in combination with the glyphosate detoxifying encoding igrA gene, recently characterized from Pseudomonas. The molecular analysis of all transgenic plant lines showed a stable integration of transgenes and their active expression in foliar tissues. The physiological analysis of glyphosate treated transgenic lines at seed germination and vegetative stages showed a significant difference in glyphosate tolerance between the two transgenic groups. The transgenic plants with OsmEPSPS and igrA genes, representing dual glyphosate tolerance mechanisms, showed an improved root-shoot growth, physiology, overall phenotype and higher level of glyphosate tolerance compared to the OsmEPSPS transgenic plants. This study highlights the advantage of igrA led detoxification mechanism as a crucial component of glyphosate tolerance strategy in combination with glyphosate tolerant OsmEPSPS gene, which offered a better option to tackle in vivo glyphosate accumulation and imparted more robust glyphosate tolerance in rice transgenic plants.
Highlights
Weeds are the most serious biological constraints in agriculture, which have enormous impact on crop plants in terms of growth, development, and grain yield
The position of amino acid proline was found conserved in all the plant enolpyruvyl shikimate 3-phosphate synthase (EPSPS) protein and found at position 182 corresponding to A. thaliana, 180 to L. multiflorum and 173 aa in rice EPSPS
The ‘S’ group transgenic lines, expressing OsmEPSPS, exhibited a comparable shoot growth, the root growth was significantly inhibited (Figures 4A–C). These results clearly suggest that co-expression of both OsmEPSPS and increased glyphosate resistance (igrA) genes confers better glyphosate tolerance and phenology compared to ‘S’ transgenic plant lines expressing only OsmEPSPS gene
Summary
Weeds are the most serious biological constraints in agriculture, which have enormous impact on crop plants in terms of growth, development, and grain yield. The in silico analysis of various plant EPSPS polypeptide sequences revealed a conserved position of proline amino acid at position 173 (173 in rice and 182 in Arabidopsis), which is replaced with serine in several glyphosate resistant weeds (Supplementary Table 1). We selected this mutation point at 173 and replaced it with serine in rice EPSPS gene by PCR based mutagenesis technique to make it glyphosate tolerant. For making expression cassette with igrA, the rice codon optimized synthetic 1,058 bp igrA coding DNA sequence was cloned under rice actin 2 promoter (2,578 bp, amplified using Act2-PF and Act2-PR primers) and its 3 terminator (223 bp, using primers Act2-TF and Act2-TR) in Gateway R compatible entry vector 2 (pL34R12-Cm-ccdB) (Chen Q.J. et al, 2006)
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