Electrical Manipulation of Cells pp 201-222
Electroporation and Transgenic Plant Production
Electroporation is a well-established method for production of transgenic plants. Short high-voltage pulses can permeabilize the protoplast plasma membrane, facilitating uptake of plasmid DNA that can become expressed transiently and, eventually, be stably incorporated into the genome.
The major electrical parameters are field strength and pulse duration, which are inversely related and can be chosen within wide ranges (100–5000 V/cm and 0.01–100 msec). Stable transformation requires less rigorous electrical conditions than transient expression. Transient and stable transformation increase with plasmid DNA concentration, up to about 100 µg/mL; addition of carrier DNA lowers the amount of plasmid DNA required for transformation. Linearized plasmid DNA and heat shock enhance stable transformation. Addition of PEG stimulates transient expression and, in most cases, stable transformation. The transformation rate is also affected by protoplast size, pulse type, culture medium, and temperature.
Stable transformation frequencies are in the range 0.0001–0.1% of the electroporated protoplasts. Transgenic plants contain, on average, from one to three copies of the exogenous gene, and all copies are usually integrated into one site in the genome. The inserted plasmid DNA is often modified by rearrangement and ligation events, and the copy number does generally not correlate with expression level. Transgenic plants regenerated from electroporated protoplasts are most often fertile, and the exogenous genes appear to be inherited as a single dominant character in a Mendelian fashion.
Although the cell wall is generally regarded to be impermeable to DNA, some intact cells and tissues can be induced to take up DNA by electroporation.
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