Aminoglycoside antibiotics inhibit shoot regeneration from Siberian elm leaf explants

Abstract

Four aminoglycoside antibiotics were evaluated for their effects on shoot regeneration from leaf explants of Siberian elm (Ulmus pumila L.) seedlings and their potential use as selective agents in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene. Kanamycin at 100 mg·L-1 or higher concentration reduced shoot regeneration, with complete inhibition at 225 mg·L-1, and was considered a suitable selective agent. Neomycin completely inhibited shoot regeneration at 450 mg·L-1, but all explants remained green; therefore, it may also be used as a selective agent. Geneticin significantly inhibited shoot formation at 1 mg·L-1 and completely killed the explants at 4 mg·L-1 after 1 week. Geneticin was too toxic for direct selection, but may be useful in a delayed selection scheme or for confirmation of transformation. Paromomycin was least effective in inhibiting shoot formation; 13% of explants still regenerated shoots on the medium with the highest concentration tested (400 mg·L-1). Both neomycin and paromomycin precipitated in media containing Phytagel as a gelling agent if antibiotic stock solutions were added to the medium without adjusting their pH. Precipitation was prevented by adjusting the pH of the stock solutions from 6.2 (neomycin) or 6.9 (paromomycin) to above 9, or by using agar as a gelling agent. The precipitation was not affected by the concentrations of salts in the media.