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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 ×domestica Borkh.) and on shoot regeneration. Exposure of leaf explants to regeneration and elongation media containing 100 μg·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%, 11%, and 50%, respectively, compared to 67% for the controls. Short-term (1- to 18-hour) vacuum infiltration with 500 μg·mL-1 of any of the above antibiotics did not inhibit regeneration and failed to eliminate A. tumefaciens from leaf explants. Cef (2000 μg·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 μg·mL-1), and a 52-day incubation on regeneration and elongation media containing 100 μg·mL-1 each of mef and crb. Kan resistant, GUS (β-glucuronidase) positive, putative transformants without A. tumefaciens were generated by adding kan (10 μg·mL-1) to the regeneration and elongation media.
Particle bombardment has been shown to be useful for genetic manipulation of many plants; however, a critical component for successful transformation is the ability of transformed cells to regenerate plants. This study describes factors that affect the regeneration efficiency of apple leaf explants following particle bombardment. Basal leaf segments of micropropagated `Royal Gala' apple were treated with 1μm gold particles (0.5 μg/10μl), accelerated at either 4.5, 6.2, 7.6, 9.3 or 13.8 MPa, and cultured on N6 salts + l0μM TDZ regeneration medium for 5, 10 or 20 days in the dark. Both microprojectile-treated and control explants exhibited 85-100% regeneration. However, only 30-60% of the explants bombarded at 7.6, 9.3 and 13.8 MPa had more than 10 regenerants and 6-10% had more than 20 regenerants, whereas for control explants and those bombarded at 4.5 and 6.2 MPa, 70-90% had more than 10 regenerants and 30-50% had more than 20.