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Zapping DNA into plant cells.

A jolt of electrical current is what it may take to get plant genetic engineering off the ground. Scientists at Boyce Thompson Institute for Plant Research (BTI) in Ithaca, N.Y., report the first success in plant cells of the technique called electro-transformation, or electroporation. In the experiments, high voltage electrical pulses opened pores of carrot cell membranes, allowing foreign DNA to enter the cells, to become incorporated into chromosomes and to be expressed. This technique has already been used in a few instances for DNA uptake by animal cells.

"The introduction of foreign genetic material in the form of 'naked' or free DNA is the fundamental requirement for genetic engineering of plants," says Aladar A. Szalay of BTI. Most current plant genesplicing employs a more elaborate procedure -- foreign genes are inserted into the plasmid, called Ti, of the bacterium, Agrobacterium tumefaciens. The bacterium is used to deliver its elaborately engineered payload and then must be destroyed. "It's a long process," says William Langridge of BTI. This procedure is limited to those plants, mostly dicotyledons (thus not the major cereal crops), that the bacterium can infect.

In contrast, electroporation could enable any gene to be directly introduced into plant cells, Langridge predicts. For their initial experiments, Langridge and Bao-Jian Li, a visiting geneticist from Cungshan University in the People's Republic of China, used the Ti plasmid and a smaller plasmid as examples of foreign DNA. The recipient wild carrot cells were treated with enzymes to remove the cell wall and expose the cell membrane. Short pulses of 40-volt direct current were applied to a 0.4-milliliter mixture of plasmid molecules and about a million of the enzyme-treated carrot cells, called protoplasts.

About 2 percent of the carrot cells took up and expressed the foreign DNA, Langridge reports. Without electroporation, only one cell in a million takes up foreign DNA molecules. For the smaller plasmid, Langridge calculates about 10 copies go into each cell. In the case of the Ti plasmid, the cells containing the plasmid regenerated into plant embryos, but then grew aberrantly due to extra hormone encoded by genes carried on the Ti plasmid.

The electroporation technique works to introduce RNA, as well as DNA, into plant protoplasts. In experiments with tobacco mosaic virus RNA, almost 80 percent of the tobacco protoplasts were infected after electrical pulses, compared to less than 3 percent in an unshocked mixture of protoplasts and the RNA. The scientists are now working to improve and simplify this "exceptionally useful" tool for plant genetics.
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Title Annotation:electrotransformation
Author:Miller, Julie Ann
Publication:Science News
Date:Jul 13, 1985
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