ANALYTICAL APPROACHES FOR QUANTIFICATION OF GMOS IN ALFALFA

The adoption of genetically modified (GM) crops varies greatly globally, with widespread use in America, Asia, Africa, and Australia, while in Europe is extremely limited. The adventitious presence of genetically modified organisms (GMOs) in conventional crops poses a challenge to the genetic purity of seeds and derived products. This study focused on the development of analytical approaches for the detection and quantification of GMOs in alfalfa (Medicago sativa), with particular reference to events MON-Ø00101-8 and MON-Ø0163-7, which confer tolerance to glyphosate. The work involved the isolation and characterization of 22 transgenic genotypes. The number of transgene copies was determined using real-time PCR and droplet-digital PCR. Using freeze-dried leaf tissue from some of the genotypes mentioned above, reference materials with known GMO content, in percentage by weight and number of copies, were developed. The quality of these materials was verified by real-time PCR with calibration curve calculation and analysis of validation samples with known GMO content. The same real-time PCR analyses conducted on three commercial seed samples with two different assays provided estimates that were in close agreement with each other. For one of the three samples, with a very high GMO content, a very similar estimate was obtained using the delta-deltaCt approach. Finally, for two of the genotypes (M-28 and M-115) useful for future experimental use as reference and validation material, a protocol for micropropagation and in vitro maintenance was developed, with significant advantages in terms of phytosanitary safety and compliance with current regulations on transgenic plants. The results of this study highlight the effectiveness of the analytical approaches adopted for the quantification of GMOs in alfalfa and support the usefulness of the reference materials developed for genetic purity testing of seeds and derived products in international trade.