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

抄録

Plastid transformation in higher plants has been established in tobacco using particle bombardment, and the protocol 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 efficiency may be increased by modification of leaf treatment to lessen such damage. In the modified protocol, tobacco leaves were cut into pieces (5×5 mm) and placed on the culture medium plate (approximately fifty pieces per plate) for one day before bombardment. After 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′-adenylyltransferase gene and a green fluorescence protein gene were used for plastid transformation. Approximately four independent plastid transformants per bombarded plate were obtained on average using the modified protocol; the transformation efficiency was 1.6 times higher than that in a control experiment using the standard protocol. Modified leaf treatment improved the efficiency and stability of plastid transformation. This finding should aid plastid transformant production in tobacco and other plant species.