Transgenic Cotton (Gossypium hirsutumL.) to Combat Weed Vagaries: Utility of an Apical Meristem-Targetedin plantaTransformation Strategy to Introgress a ModifiedCP4-EPSPSGene for Glyphosate Tolerance

Weeds burden plant growth as they compete for space, sunlight, and soil nutrients leading to 25-80% yield losses. Glyphosate [N-(phosphonomethyl)glycine] is a widely used broad spectrum non-selective herbicide that controls weeds by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme and interfering with the shikimate biosynthesis pathway. Cotton (Gossypium hirsutumL.) is one of the most important commercial crops grown worldwide for its fiber. We have developed herbicide tolerant transgenic cotton (cv. P8-6) by introgression of a codon-optimized and modifiedEPSPSgene (CP4-EPSPS) possessing an N-terminal chloroplast targeting peptide fromPetunia hybrida. Because of the recalcitrant nature of cotton, a genotype-independent non-tissue culture-based apical meristem-targetedin plantatransformation approach was used to develop transformants. Althoughin plantatransformation methodologies are advantageous in developing a large number of transgenic plants, effective screening strategies are essential for initial identification of transformants. In the present study, the use of a two-level rigorous screening strategy identified 2.27% of T1 generation plants as tolerant to 800 and 1,500 mg/L of commercially available glyphosate (Roundup). Precise molecular characterization revealed stable integration, expression, and inheritance ofCP4-EPSPSin advanced generations of the promising transgenic events. Further, superiority of selected transgenic plants in tolerating increasing levels of glyphosate (500-4,000 mg/L) was ascertained through reduced accumulation of shikimate. This report is the first of its kind where cotton transformants tolerating high levels of glyphosate (up to 4,000 mg/L) and accumulating low levels of shikimate have been identified. This study not only reiterated the genotype-independent nature of the transformation strategy but also reiterated the translational utility of theCP4-EPSPSgene in management of weeds.