Improvement of the plastid transformation protocol by modifying tissue treatment at pre- and post-bombardment in tobacco

Abstract

Plastid transformation in higher plants has been established in tobacco using particle bombardment, and the p rotocol has been used as a model in other plant species; however, as target materials, tobacco leaves may be partially exposed to unexpected abiotic stresses—drought stress during post-bombardment culture and damage due to cutting before selection culture. Plastid transformation eciency may be increased by modication of leaf treatment to lessen such d amage. In the modied protocol, tobacco leaves were cut into pieces (5×5 mm) and placed on the culture medium plate (approximately ��y pieces per plate) for one day before bombardment. Aer bombardment, these pieces were transferred onto the non-selection medium and cultured for three days; they were then transferred onto spectinomycin selection culture medium. A transformation vector containing an aminoglycoside 3�-adenylylt ransferase gene and a greenuorescence protein gene were used for plastid transformation. Approximately four independent plastid transformants per bombarded plate were obtained on average using the modied protocol; the transformation eciency was 1.6 times higher than that in a control experiment using the standard protocol. Modied leaf treatment improved the eciency and stability of plastid t ransformation. �isnding should aid plastid transformant production in tobacco and other plant species.