An intragenic approach to confer glyphosate resistance in chile (Capsicum annuum) by introducing an in vitro mutagenized chile EPSPS gene encoding for a glyphosate resistant EPSPS protein

Abstract

Chile pepper (Capsicum annuum) is an important high valued crop worldwide, and when grown on a large scale has problems with weeds. One important herbicide used is glyphosate. Glyphosate inactivates the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the synthesis of aromatic amino acids. A transgenic approach towards making glyphosate resistant plants, entails introducing copies of a gene encoding for glyphosate-resistant EPSPS enzyme into the plant. The main objective of our work was to use an intragenic approach to confer resistance to glyphosate in chile which would require using only chile genes for transformation including the selectable marker. Tobacco was used as the transgenic system to identify different gene constructs that would allow for the development of the intragenic system for chile, since chile transformation is inefficient. An EPSPS gene was isolated from chile and mutagenized to introduce substitutions that are known to make the encoded enzyme resistant to glyphosate. The promoter for EPSPS gene was isolated from chile and the mutagenized chile EPSPS cDNA was engineered behind both the CaMV35S promoter and the EPSPS promoter. The leaves from the transformants were checked for resistance to glyphosate using a cut leaf assay. In tobacco, though both gene constructs exhibited some degree of resistance to glyphosate, the construct with the CaMV35S promoter was more effective and as such chile was transformed with this gene construct. The chile transformants showed resistance to low concentrations of glyphosate. Furthermore, preliminary studies showed that the mutated EPSPS gene driven by the CaMV35S promoter could be used as a selectable marker for transformation. We have shown that an intragenic approach can be used to confer glyphosate-resistance in chile. However, we need a stronger chile promoter and a mutated chile gene that encodes for a more glyphosate resistant EPSPS protein.

Genome Editing and Improvement of Abiotic Stress Tolerance in Crop Plants

“…The advancement of glyphosate-resistant plants requires changes in the machinery of some genes [203]. 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme is implicated in the formation of aromatic compounds in crops with the transfer of phosphoenolpyruvate (PEP) enzyme for activating the reaction [203,273]. Glyphosate hinders the act of the EPSPS enzyme by inhibiting the add-on of glyphosate to the PEP enzyme binding sites, eventually blocking the formation of aromatic products and causing crop death [203].…”

Plant-associated and soil microbiota composition as a novel criterion for the environmental risk assessment of genetically modified plants

“…Genetic transformation technologies (GTTs) are used to manipulate GM plants to provide them with beneficial properties, such as drought-resistance or protection from weeds. 46,47 Besides antibiotic resistance genes, which carry the potential risk of dissemination of antibiotic resistance in ecosystems, [48][49][50] GTTs are based on selectable markers and employ strategies which include the removal of the antibiotic marker genes from transgenic plants. 51 The markers lysine racemase, 52 phosphomannose isomerase, 53,54 and sulfadiazine 55 are currently in use or have beensuggested for use in plant GTTs.…”

Production of EPSPS and bar gene double-herbicide resistant castor (Ricinus communis L.)

“…The EPSPS and Bar genes have been introduced into pepper, soybean, maize, millet, potato, and other crops, and they have yielded high glyphosate- [16,17] and glufosinate- [18,19] resistance. Herbicide resistance among weeds is inevitable, and long-term use of a single herbicide will spread herbicide resistance.…”