Production of herbicide-resistant coffee plants (Coffea canephora P.) via Agrobacterium tumefaciens-mediated transformation

Abstract

Transgenic plants of Coffea canephora P. resistant to the herbicide ammonium glufosinate were regenerated from leaf explants after co-culture with Agrobacterium tumefaciens strain EHA105 harboring pCambia3301, a plasmid that contains the bar and the uidA genes both under control of 35S promoter. Direct somatic embryogenesis was induced on basal medium contained 1⁄4 strength macro salts and half strength micro salts of MS medium, organic constituents of B 5 medium and 30 g.L -1 sucrose supplemented with 5 μ M N 6 – (2-isopentenyl)-adenine (2-iP). Ten μ M ammonium glufosinate was used for putative transgenic somatic embryos selection. Presence and integration of the bar gene were confirmed by PCR and Southern blot analysis. Selected transgenic coffee plants sprayed with up to 1600 mg.L -1 of Finale  , a herbicide containing glufosinate as the active ingredient, retained their pigmentation and continued to grow normally during ex vitro acclimation.Plantas transgênicas de Coffea canephora P resistentes ao herbicida glufosinato de amônio foram regeneradas a partir de explantes foliares co-cultivados com Agrobacterium tumefaciens EHA105 contendo o plasmídio pCambia3301 que contém os genes bar e uidA ambos sob controle do promotor 35S. Embriogênese somática direta foi induzida no meio contendo 1⁄4 da concentração de macro, metade da concentração de micronutrientes do meio MS, constituintes orgânicos do meio B 5 e 30 g.L -1 de sacarose suplementado com 5μM N 6 – (2-isopentenil)-adenina (2-iP) e 10 μM de glufosinato de amônio para seleção de embriões transgênicos putativos. A presença e a integração do gene bar foram confirmados pelas análises de PCR e Southern blot. As plantas transgênicas selecionadas de café, pulverizadas com 1600 mg.L -1 do herbicida Finale que contém glufosinato como ingrediente ativo, mantiveram a coloração e continuaram crescendo normalmente na aclimatação ex vitro.