Articles
GENERATING APPLE TRANSFORMANTS FREE OF AGROBACTERIUM TUMEFACIENS BY VACUUM INFILTRATING EXPLANTS WITH AN ACIDIFIED MEDIUM AND WITH ANTIBIOTICS
Article number
530_10
Pages
103 – 112
Language
Abstract
As part of a program to develop transgenic ‘Royal Gala’ apple (Malus x domestica Borkh), long-term exposure to antibiotics was compared with short-term vacuum infiltration with an acidified (pH 3.0) medium and/or a range of antibiotics for their effect on shoot regeneration and on eliminating Agrobacterium tumefaciens, supervirulent strain EHA101, from apple leaf explants following co-cultivation.
None of the antibiotics, carbenicillin (crb), cefotaxime (cef) or mefoxin (mef), when incorporated into regeneration medium at 100 µg ml-1 eliminated A. tumefaciens from explants and cef inhibited shoot regeneration.
Vacuum infiltration with either crb, cef, or mef at 2000 µg ml-1 for 30 min did not inhibit shoot regeneration; however, none of the treatments eliminated A. tumefaciens from explants.
Vacuum infiltration with either acidified medium for 1 h or cef (5000 µg ml-1) for 18 h reduced contamination by about 20 – 30 %, whereas combining the two treatments reduced contamination with A. tumefaciens by 87 %. Kanamycin (kan) resistant, putative transformants without A. tumefaciens were generated when explants, following a 2-d co-cultivation with strain EHA101, were vacuum infiltrated for 1 h with an acidified medium, followed by an 18 h vacuum infiltration with cef at 5000 µg ml-1 and incubated for 52 days on regeneration and elongation media with crb (100 µg ml-1), mef (100 µg ml-1), and kan (10 µg ml-1).
None of the antibiotics, carbenicillin (crb), cefotaxime (cef) or mefoxin (mef), when incorporated into regeneration medium at 100 µg ml-1 eliminated A. tumefaciens from explants and cef inhibited shoot regeneration.
Vacuum infiltration with either crb, cef, or mef at 2000 µg ml-1 for 30 min did not inhibit shoot regeneration; however, none of the treatments eliminated A. tumefaciens from explants.
Vacuum infiltration with either acidified medium for 1 h or cef (5000 µg ml-1) for 18 h reduced contamination by about 20 – 30 %, whereas combining the two treatments reduced contamination with A. tumefaciens by 87 %. Kanamycin (kan) resistant, putative transformants without A. tumefaciens were generated when explants, following a 2-d co-cultivation with strain EHA101, were vacuum infiltrated for 1 h with an acidified medium, followed by an 18 h vacuum infiltration with cef at 5000 µg ml-1 and incubated for 52 days on regeneration and elongation media with crb (100 µg ml-1), mef (100 µg ml-1), and kan (10 µg ml-1).
Publication
Authors
F.A. Hammerschlag, Q. Liu, R.H. Zimmerman, P. Gercheva
Keywords
antibiotics, GUS assay, low pH, Malus x domestica, transformation
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