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The genetic transformation of Simarouba glauca will help researchers across the globe in developing high medicinal value and high biomass varieties in a short period. Although genetic transformation methods have been trailed by many research groups for several years, the variation in transformation efficiency and difficulties in handling tissue culture plantlets made it difficult to optimize a reliable protocol. In this study, we established an alternative protocol of genetic transformation by introducing GUS Plus reporter gene into the genome of a S. glauca through the Agrobacterium-mediated gene transfer method which is the first report in India. Leaf explants co-cultivated with Agrobacterium LBA4404 strains harboring pCAMBIA 1305.1 vector have shown the presence of GUS Plus and HptII gene by PCR analysis as well as GUS assay. The outcome of our research provides fundamental information on efficient gene transformation in S. glauca which will help to enhance future research on the genetic transformation of S. glauca.