Optimal concentration of selective agents for inhibiting in vitro growth of Urochloa brizantha embryogenic calli

Abstract

The ability to distinguish transgenic cells of untransformed cell mass is a key step for the production of transgenic plants. Thus, the use of selection marker genes for identification of genetically modified plants is necessary. The aim of this study was to determine the optimal concentration of four selective agents (kanamycin, hygromycin, phosphinothricin and mannose) to inhibit in vitro growth of Urochloa brizantha cv. Marandu calli. Embryogenic calli were obtained from mature seeds inoculated in MS medium supplemented with 30 g/L sucrose, 3 mg/L 2,4 dichlorophenoxy acetic acid (2,4-D) and 300 mg/L hydrolysate casein and their growth rate was monitored for 74 days by measuring calli fresh weight. It was demonstrated that U. brizantha calli are more sensitive to low concentrations of hygromycin than kanamycin (25 and 50 mg/L, respectively). For the herbicide phosphinothricin, 5 mg/L was enough to prevent the calli growth, but allowed escape. Mannose should be used as the only carbon source on the plant tissue culture medium. All selective agents tested here, in the appropriate concentration, could be used in experiments aiming to produce transgenic signal grass. However, mannose selection might reduce environmental concerns about gene flow and development of herbicide resistance in escaped Urochloa populations.

Keywords: Signal grass, transformation, marker genes, selection