Susceptibility of embryogenic and organogenic tissues of maritime pine (Pinus pinaster) to antibiotics used in Agrobacterium-mediated genetic transformation

Abstract

The effects of antibiotics commonly used in Agrobacterium-mediated transformation were studied on Pinus pinaster tissues. Embryogenic tissue growth from three embryogenic lines and adventitious bud induction from cotyledons from three open-pollinated seed families were analysed. Cefotaxizme, carbenicillin and timentin commonly used for Agrobacterium elimination, at concentrations of 200–400 mg l ⁻¹ did not inhibit the embryogenic tissue growth on filter paper nor as clumps. Adventitious bud induction and bud number were significantly reduced for one of the tested families when using 400 mg l⁻¹ cefotaxime or timentin. The selection agent kanamycin significantly inhibited growth of embryogenic tissue on filter paper in all the embryogenic lines␣and concentrations tested (20–50 mg l⁻¹). Kanamycin also inhibited growth of embryogenic clumps after two subcultures at 5–50 mg l⁻¹. In␣cotyledons, kanamycin inhibited adventitious bud␣formation in the three seed families used, regardless of the concentrations tested (5–25 mg l⁻¹). There was a significant effect of the seed family on the bud induction and the number of adventitious buds produced. From the results obtained, we propose the use of timentin to eliminate Agrobacterium in transformation experiments, at concentrations of 400 mg l⁻¹ for embryogenic tissues and of 300 mg l⁻¹ for cotyledons. For selection of transformed tissues carrying the kanamycin resistance gene, kanamycin should be used at 20 mg l⁻¹ for embryogenic tissues on filter paper, at 5 mg l⁻¹ when clumps are in direct contact with the selection medium, and bellow 5 mg l⁻¹ for adventitious bud induction.