AN EFFICIENT AGROBACTERIUM-MEDIATED GENETIC TRANSFORMATION SYSTEM IN LENTIL (Lens culinaris MEDIK)

Abstract

Up to date, the transformation frequency in lentil (Lens culinaris Medik) is low. Therefore, the main objective of this study was to optimize an in vitro regeneration system compatible with an Agrobacterium-mediated transformation and to establish the best antibiotic concentration to allow the selection of transformed plants, to develop transgenic lentil plants with higher efficiency. Cotyledonary node explants were transformed with Agrobacterium tumefaciens strain GV2260 harboring binary vector pBI121 with neomycin phosphotransferase (npt-II) and ?-glucuronidase (gus) genes. A 50 mg L?1 kanamycin continuous selection regime was efficient for transformants development as did not affect shoots and roots regeneration in most explants and no escape was observed. Stable integration and expression of transgenes in lentil genome were confirmed by PCR analysis and histochemical assay for ?-glucuronidase (GUS) enzymatic activity. Wounding of explants, use of an optimized in vitro regeneration protocol, application of high acetosyringone concentration and bacterial density were important to achieve high transformation frequency. Transgenic lentil shoots were produced with an overall frequency of 7%.