Freedom to operate analysis, design and evaluation of expression cassettes that confer tolerance to glyphosate

Abstract

Tolerance to the herbicide glyphosate is the most extended feature in commercial transgenic events released worldwide and it is an example of the successful use of genetic modification to improve weed control in crops. Glyphosate-tolerant genotypes have been developed by multinational corporations using patent- protected technologies. For some of these events, all associated patents have expired and, therefore, have become a good target for the development of herbicide-tolerant agbiogenerics by national research institutions, using local crop varieties. As a first step in this process, we present the design (in silico) of three expression cassettes with the purpose of using them in the transformation of Colombian soybean (Glycine max) varieties to confer them tolerance to glyphosate. We transformed Nicotiana benthamiana as a model to validate the functionality of the expression cassettes and detected the expression of the transgene by RT-PCR. Additionally, a Freedom to Operate analysis of the sequences used in the expression cassettes suggests that their commercial use in Colombia does not infringe third party rights. This analysis must be updated and validated by intellectual property experts prior to commercialization.

Agbiogeneric soybean with glyphosate tolerance: Genetic transformation of new Colombian varieties

“…Two different binary plasmids derived from pCAMBIA 1301 were used (Jiménez-Barreto et al 2020), both from which the Hygromycin resistance gene was removed and in which the E-IGP expression casette was included. This expression casette inlcudes the soybean polyubiquitin promoter (GmUbi), the Chloroplast Transit Peptide (CTP) from Petunia hybrida, a version of the cp4epsps gene optimized for expression in soybean and the Nopaline Synthase (NOS) terminator sequence (Jiménez-Barreto et al 2020). One of the plasmids (pGEIGP) carried the uidA (GUS) gene originally included in the binary vector and the other had this gene removed (pEIGP).…”