Agrobacterium-mediated Transformation of Tomato (Lycopersicon esculentum var. Pusa Ruby) with Coat-protein Gene of Physalis Mottle Tymovirus

Abstract

Transgenic tomato plants harboring the coat protein (CP) gene of Physalis mottle tymovirus (PhMV) were obtained through Agrobacterium-mediated genetic transformation. The cotyledonary leaves were transformed by cocultivation with Agrobacterium strain LBA4404 carrying the binary vector pBI 121. The CP gene of PhMV was cloned into the binary vector by deleting the GUS gene. Cotyledonary explants were cultured on the regeneration medium supplemented with 50 mg/L kanamycin and 400 mg/L cefotaxime (MS 1). Shoots regenerated via callus. Putative transformants were transferred to a rooting medium containing 25 mg/L kanamycin (MS2), where successful rooting was obtained. Transgenic nature of the transformants was checked by PCR and Western analyses. The T[1] progenies segregate into a 3 :1 ratio. Southern analysis confirmed the integration of CP gene into the tomato genome of T[1] progenies. The transgenic tomato plants were further challenged with PhMV and exhibited delay in symptom development, thus confirming the establishment of transgenic plants with partial resistance to the virus