Factors affecting gene transfer efficiency to apricot leaves duringearly Agrobacterium-mediated transformation steps

Abstract

A procedure to transfer marker genes to apricot leaves has been developed by assessment of different factors affecting early Agrobacterium-mediated transformation steps. Three oncogenic strains (C58, Ach5 and A281) were equally efficient in inciting tumours in apricot seedlings, however two disarmed derivatives from those strains (C58/pMP90 and EHA105) differently transformed apricot leaves in vitro. Maximum transient gfp expression was found 7-9 d after infection and stable expression could be determined four weeks later. An optimized procedure, based on transformation efficiencies (measured as percentage of transformed explants and average gfp spots per transformed explant) and control of the Agrobacterium growth after co-culture, was established. It consisted of infecting leaves for 10 min in a bacterial suspension (cultured for 24 h in a simplified medium with 500 μM AS and diluted to about 107 cfu ml-1), with a co-culture time of 4 d in the presence of l00 μM acetosyringone.With these conditions all explants were transformed and more than 20 gfp spots per explant, as average, were recorded. Calli expressing gfp and resistant to 172 μM kanamycin have been obtained and maintained in culture for more than one year. Amplification of both transgenes was also verified by PCR and its integration by southern blot analysis. This is, to our knowledge, the first report on transformation of apricot leaves with an adult origin.