Colorimetric and Chlorophyll Fluorescence Assays for Fast Detection and Selection of Transgenic Events of Cotton, Cowpea, Soybean and Common Bean Expressing the Atahas Gene

The detection of the presence and expression of transgenes in genetically modified plants is a key step in the process ofselecting promising lines. We adapted two methods developed for detecting tolerance to the herbicide imazapyr for selection oftransgenic lines expressing the mutated acetohydroxyacid synthase enzyme (AHAS) from Arabidopsis thaliana (Atahas gene). Thiswas achieved using transgenic events from cotton, cowpea, soybean and common bean, which have previously been transformed toexpress the mutated Atahas gene. In the first method, a colorimetric assay was developed that detects acetoin, an intermediate in thebiosynthetic pathway of branched chain amino acids, which is accumulated in the presence of cyclopropanedicarboxylic acid (CPCA),an inhibitor of ketoacid reductoisomerase (KARI). In the presence of the herbicide, it was possible to distinguish non-transgenic fromtransgenic plants. Qualitative analysis of acetoin formed during the AHAS inhibition allowed to indirectly determine the Atahastransgene expression. The second method measured the kinetics of chlorophyll fluorescence emission. Leaf discs pre-treated withimazapyr for 24 hours were evaluated using the modulated fluorimeter for maximum quantum efficiency of Photosystem II (PSII)(Fv/Fm) and relative electron transport rate (ETR). Results showed that almost all species analyzed presented a marked decrease inFv/Fm after treatment with imazapyr. In addition, the ETR was significantly reduced in transgenic plants treated with the herbicide. Collectively, our results showed that it is possible to identify transgenic plants expressing Atahas gene and infer their levels of toleranceto imazapyr at a very early stage after transformation.