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

摘要

The effects of antibiotics commonlyused in Agrobacterium-mediated transformationwere studied on Pinus pinaster tissues. Embryogenictissue growth from three embryogenic linesand adventitious bud induction from cotyledonsfrom three open-pollinated seed families wereanalysed. Cefotaxizme, carbenicillin and timentincommonly used for Agrobacterium elimination, atconcentrations of 200–400 mg l –1 did not inhibitthe embryogenic tissue growth on filter paper noras clumps. Adventitious bud induction and budnumber were significantly reduced for one of thetested families when using 400 mg l–1 cefotaximeor timentin. The selection agent kanamycinsignificantly inhibited growth of embryogenictissue on filter paper in all the embryogeniclines and concentrations tested (20–50 mg l–1).Kanamycin also inhibited growth of embryogenicclumps after two subcultures at 5–50 mg l–1.In cotyledons, kanamycin inhibited adventitiousbud formation in the three seed families used,regardless of the concentrations tested (5–25 mg l–1). There was a significant effect of theseed family on the bud induction and the numberof adventitious buds produced. From the resultsobtained, we propose the use of timentin toeliminate Agrobacterium in transformationexperiments, at concentrations of 400 mg l–1 forembryogenic tissues and of 300 mg l–1 for cotyledons.For selection of transformed tissues carryingthe kanamycin resistance gene, kanamycinshould be used at 20 mg l–1 for embryogenic tissueson filter paper, at 5 mg l–1 when clumps arein direct contact with the selection medium, andbellow 5 mg l–1 for adventitious bud induction.