Effect of antibiotics and exposure to an acidified medium on the elimination of Agrobacterium tumefaciens from apple leaf explants and on shoot regeneration

A range of antibiotics and short-term exposure to an acidified (pH 3.0) medium were evaluated for their effects on eliminating Agrobacterium tumefaciens, supervirulent strain EHA101 (pEHA101/pGT100), from leaf explants of 'Royal Gala' apple (Malus xdomestica Borkh,) and on shoot regeneration, Exposure of leaf explants to regeneration and elongation media containing 100 mu g.mL(-1) concentrations of the antibiotics carbenicillin (crb), cefotaxime (cef), and cefoxitin [=mefoxin (mef)], singly or in combination for 52 days did not eliminate A. tumefaciens from the explants, The percentage of regeneration on crb, cef, and mef was 97 %, ii %, and 50 %, respectively, compared to 67 % for the controls. Short-term (1- to ib-hour) vacuum infiltration with 500 mu g.mL(-1) of any of the above antibiotics did not inhibit regeneration and failed to eliminate A. tumefaciens from leaf explants, Cef(2000 mu g.mL(-1)) did not inhibit the percentage of regeneration and was more effective than crb or mef in preventing growth of A. tumefaciens when vacuum infiltrated into apple leaf explants for 30 minutes, Further experiments demonstrated that the incidence of A. tumefaciens contamination could be reduced to 28% without negatively impacting shoot regeneration by using a 1-hour vacuum infiltration with an acidified medium, an 18-hour vacuum infiltration with cef(5000 mu g.mL(-1)), and a 52-day incubation on regeneration and elongation media containing 100 mu g.mL(-1) each of mef and crb, Kan resistant, GUS (beta-glucuronidase) positive, putative transformants without A. tumefaciens were generated by adding kan (10 mu g.mL(-1)) to the regeneration and elongation media.