Direct gene transfer to plant protoplasts by mild sonication.
A novel procedure employing mild sonication for transformation of plant protoplasts is described. Transient expression of a chloramphenicol acetyltransferase (CAT) gene in protoplasts of sugar beet (Beta vulgaris L.) and tobacco (Nicotiana tabacum L.) was obtained by a brief exposure of the protoplasts to 20 kHz ultrasound in the presence of plasmid DNA. Maximum levels of CAT activity were achieved by sonication for 500-900 ms at 30-70 W electric power (0.65-1.6 W/cm2 acoustic power). This reduced the viability to 15-20 % and 60 % for sugar beet and tobacco protoplasts, respectively. Up to 12 % (sugar beet) and 81 % (tobacco) of maximum transient expression could be achieved with no significant loss of viability. Protoplasts surviving exposure to ultrasound were found to have a similar long-term viability and to regenerate to micro-calli as untreated protoplasts. Plasmid DNA concentrations of 80-110 μg/ml and sucrose concentrations of 21-28 % in the sonication medium were found to be optimal for transient expression.
Biotechnology Research Division, DANISCO A/S, Langebrogade 1, DK-1001, Copenhagen K, Denmark.
M
M
Joersbo
J
J
Brunstedt