Genetic transformation of green bean callus via Agrobacterium mediated DNA transfer

Abstract

Kanamycin resistant callus was produced from leaf disc or hypocotyl expiants of green bean (Phaseolus vulgaris L.) when cultured on a defined medium containing 50 mg/l kanamycin after 4 days of co-cultivation with Agrobacterium tumefaciens strain EHA101 containing the binary vector pKYLX71GUS. The presence of neomycin phosphotransferase II (NPT-II) in crude cellular extracts from the kanamycin resistant callus was confirmed by ELISA. The expression of the ß-glucuronidase (GUS) reporter gene was confirmed by histochemical and fluorimetric analyses. Southern blot border analysis confirmed the integration of the foreign DNA. In addition to the evidence obtained from Southern analysis, the absence of Agrobacterium in the transformed callus cultures was confirmed by microscopic observation and through test cultures. Using the above protocol, bean callus cultures were also transformed with a bean chalcone synthase promoter-GUS fusion. These cultures, when treated with the elicitor glutathione, showed higher levels of GUS expression than the unelicited callus clumps.