Submitted to: Acta Horticulture Proceedings
Publication Type: Proceedings
Publication Acceptance Date: August 25, 1999
Publication Date: September 20, 2000
Citation: Hammerschlag, F.A., Liu, Q., Zimmerman, R.H., Gercheva, P. 2000. Generating apple transformants free of agrobacterium tumefaciens by vacuum infiltrating with an acidified medium and with antibiotics. Acta Horticulture Proceedings.530:103-108
Interpretive Summary: Conventional breeding of apple is a slow, lengthy process due to its long juvenile period, high level of self-incompatibility and the highly heterozygous nature of the genome. Although gene transfer techniques make it possible to overcome these problems by direct transfer of useful genes (e.g. for disease and pest resistance) into commercially important cultivars, very few transgenic apple trees have been produced. A limiting factor has been the inability to use highly virulent strains of the bacterium, Agrobacterium tumefaciens, necessary for high frequency transformation because: 1) these strains are difficult to eliminate from plant tissues after transformation, and 2) high levels of antibiotics required to eliminate A. tumefaciens from plant tissues inhibit plant regeneration. In this study, we describe a system for eliminating A. tumefaciens from apple leaf explants without interfering with plant regeneration. This research should be of use to scientists interested in using Agrobacterium-mediated gene transfer to introduce useful genes into apple and related species.
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 ug per ml eliminated A. tumefaciens from explants and cef inhibited shoot regeneration. Vacuum infiltration with either crb, cef, or mef at 2000 ug per 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 per 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 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 ug per ml and incubated for 52 days on regeneration and elongation media with crb (100 ug per ml), mef (100 ug per ml), and kan (10 ug per ml).