Transgenic Cotton (Gossypium hirsutum L.) to Combat Weed Vagaries: Utility of an Apical Meristem-Targeted in planta Transformation Strategy to Introgress a Modified CP4-EPSPS Gene for Glyphosate Tolerance

Abstract

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 5enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme and interfering with the shikimate biosynthesis pathway. Cotton (Gossypium hirsutum L.) 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 modified EPSPS gene (CP4-EPSPS) possessing an N-terminal chloroplast targeting peptide from Petunia hybrida. Because of the recalcitrant nature of cotton, a genotype-independent non-tissue culture-based apical meristem-targeted in planta transformation approach was used to develop transformants. Although in planta transformation 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 of CP4-EPSPS in 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 the CP4-EPSPS gene in management of weeds.

An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation

“…In planta transformation strategies aid in the development of transgenic plants by completely circumventing tissue culture steps and have cardinal contributions in plant biotechnology ( Kesiraju and Sreevathsa, 2017 ; Karthik et al, 2020 ). Despite efforts toward demonstration of the non-tissue culture based transformation in flax ( Bastaki and Cullis, 2014 ; Maher et al, 2020 ; Majumder et al, 2020 ; Tyagi et al, 2020 ; Zlobin et al, 2020 ), development of large-scale stable transgenics for functional genomics study in flax is still lacking.…”

“…Cardinal contributions in the field of crop improvement, especially in case of those crops that are difficult to regenerate, have been devised by deploying tissue culture independent in planta transformation methodologies ( Kesiraju and Sreevathsa, 2017 ; Karthik et al, 2020 ). Innumerable strategies that target T-DNA to discrete parts of plants and avoid tissue culture-induced somaclonal variations have emerged to aid genetic engineering of crop plants ( Kaur and Sugani, 2019 ).…”

Exploitation of Novel Bt ICPs for the Management of Helicoverpa armigera (Hübner) in Cotton (Gossypium hirsutum L.): A Transgenic Approach

“…Owing to the recalcitrance of cotton to regeneration, transgenic plants were generated by targeting the desired genes individually to the shoot apical meristem following an in planta transformation strategy ( Karthik et al, 2020a ; Kesiraju et al, 2020 ). A total of 4,600 seedlings of cotton cultivar P8–6 were subjected to Agrobacterium transformation independently for cry1AcF and cry2Aa .…”

“…The bacterial culture was later re-suspended in 100 mL of Winans’ AB medium (pH 5.2) and incubated for 18 h at 28°C. Two-day old seedlings were used for in planta transformation using the standardized protocol ( Karthik et al, 2020a ; Kesiraju et al, 2020 ). The primary transformants were transferred to the greenhouse and allowed to set seeds.…”

“…In the present study, we demonstrate the utility of two Bt ICPs, cry1AcF , and cry2Aa for the management of H. armigera in transgenic cotton. Considering the recalcitrant nature of cotton to regeneration by tissue culture, utilization of an apical meristem targeted in planta transformation for the development of stable events is an added advantage ( Kesiraju and Sreevathsa, 2017 ; Karthik et al, 2020a ; Kesiraju et al, 2020 ). Our results reconfirmed both the amenability of cotton to in planta transformation strategy as well as efficacy of the two cry toxin genes toward the management of the devastating cotton bollworm, H. armigera .…”