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United States Department of Agriculture

Agricultural Research Service

Title: Generating Apple Transformants Free of Agrobacterium Tumefaciens by Vacuum Infiltrating Explants with An Acidified Medium and with Antibiotics

item Hammerschlag, Freddi
item Liu, Qingzhong - CONTRACT EMPLOYEE
item Zimmerman, Richard
item Percheva, Petya - CONTRACT EMPLOYEE

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: August 25, 1999
Publication Date: N/A

Technical 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 eliminating Agrobacterium tumefaciens, supervirulent strain EHA101, from apple leaf explants following cocultivation and on shoot regeneration. None of the antibiotics carbenicillin (crb), cefotaxime (cef) or mefoxin(mef), when incorporated into regeneration medium at 100 ug/ ml, eliminated A. tumefaciens from explants, and cef inhibited shoot regeneration. Vacuum infiltration with either crb, cef, or mef at 500 ug/ml for 1 or 18 h, or 2000 ug/ ml 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 ug/ml) for 18 h reduced contamination by about 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 cocultivation with strain EHA101, were vacuum infiltrated for 1 h with an acidified medium, followed by an 18-h vacuum infiltration with cef at 5000 ug/ml and 52 days on regeneration medium with crb (100 ug/ml), mef (100 ug/ml), and kan (10 ug/ml).

Last Modified: 4/22/2015
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