The effect of antibiotics on elongation and callus and bud formation from embryonic tissue of Piceaglauca

Abstract

Strategies for the selection of transformed cells and the counterselection (clearing) of Agrobacterium in genetic engineering requires a balance between the selection pressures and the normal morphogenetic response of transformed tissue. In this paper, we report the effect of antibiotics used for plant transformation studies on the elongation and induction of buds from embryos and the induction of callus from seedling tissue of Piceaglauca. Carbenicillin, ampicillin (500 and 750 μg/mL), and cefotaxime (250 and 500 μg/mL) are used to clear the invitro environment of bacteria and have little effect on the elongation or bud formation from P. glauca embryos. Concentrations of hygromycin greater than 1 μg/mL and kanamycin greater than 10 μg/mL inhibited elongation, whereas concentrations of kanamycin greater than 5 μg/mL totally inhibited adventitious bud formation, callus formation, and caused seedling mortality. At concentrations as low as 1 μg/mL, kanamycin decreased adventitious bud formation by greater than 50%. These results suggest that effective selection of transformants expressing marker genes can be performed using relatively low concentrations of kanamycin or hygromycin.

Genetic transformation of Norway spruce (Picea abies (L.) Karst) using somatic embryo explants by microprojectile bombardment

“…These levels of inhibition are similar to that reported for white spruce [13] and lobloUy pine [33].…”

“…Transformed line 2-33 segregated from embryogenic tissue, which was not transformed. Kanamycin selection, which has been shown to affect organogenesis [13], may have had a deleterious effect on embryogenesis in this system. ence of one major Hind III band hybridizing with gusA (lane 11) shows that multiple intact copies of this gene, which was not selected for, are present.…”

Agrobacterium-mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants

“…We tested the effect of cefotaxime and found that the inhibitory effect of cefotaxime was roughly linear for concentrations up to 500 mg 1-1. These results were unanticipated because concentrations as high as 500 mg 1-1 were reported to have little effect on plant tissues (Pollock et al 1983;Ellis et al 1989;Tsang et al 1989). When we reduced the concentration of cefotaxime from 500 mg 1-1 tO 250 mg 1-1, callus growth was acceptable and growth of residual Agrobacterium was not a problem.…”

“…Although we attempted to simplify the procedure by plating the suspension cultures directly to shoot induction medium, this approach was unsuccessful. Second, antibiotics tend to inhibit callus growth less effectively than they inhibit organogenesis (Ellis et al 1989), potentially making selection more difficult. Finally, although our transgenic plants appeared normal, the introduction of somaclonal variation may be more likely using suspension cultures, and regeneration of plants from callus is not certain.…”

SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation

“…Under selection pressure, Agrobacterium cells could gain mutations that are resistant to the carbenicillin, which results in the overgrowth of Agrobacterium . Most antibiotics are generally expensive and may negatively affect plant cell differentiation (Ellis et al, 1989; Yu et al, 2001; Li and Qu, 2011). Therefore, a more reliable and cost-effective method to inhibit the overgrowth of Agrobacterium tumefaciens is highly desirable.…”