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 ca rbenicillin (crb), cefotaxime (cef), and cefoxitin [=mefoxin (mef)], s ingly or in combination for 52 days did not eliminate A. tumefaciens f rom the explants, The percentage of regeneration on crb, cef, and mef was 97 %, ii %, and 50 %, respectively, compared to 67 % for the contr ols. 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 fa iled to eliminate A. tumefaciens from leaf explants, Cef(2000 mu g.mL( -1)) did not inhibit the percentage of regeneration and was more effec tive than crb or mef in preventing growth of A. tumefaciens when vacuu m infiltrated into apple leaf explants for 30 minutes, Further experim ents demonstrated that the incidence of A. tumefaciens contamination c ould 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 inc ubation on regeneration and elongation media containing 100 mu g.mL(-1 ) each of mef and crb, Kan resistant, GUS (beta-glucuronidase) positiv e, putative transformants without A. tumefaciens were generated by add ing kan (10 mu g.mL(-1)) to the regeneration and elongation media.