Currently plant transformation with foreign genes may be accomplished by several proven methods, including bacteriamediated transfer, microparticle bombardment, electroporation, microinjection, sonication, and chemical treatment.
By far, the most often utilized method of plant transformation involves the use of naturally occurring plant pathogenic bacteria from the genus
Agro bacterium in nature, this bacterium infects plants and transfers some of its own bacterial DNA into the plant. Through the action of proteins produced by the bacteria, bacterial DNA is made to integrate permanently into the plant’s own genomic DNA. Expression of the bacterial DNA in the plant causes the plant to produce unusual quantities of plant hormones and other compounds, called opines, which provide food for the bacteria. The unusual quantities of plant hormones around the infection site cause the plant cells to grow abnormally, producing characteristic tumors. Scientists have harnessed this pathogenic bacterium to insert genes into plants by deleting the bacterial genes that cause tumors in the plant and then inserting desirable genes in their place. When the modified Agrobacterium infects a plant, it transfers the desir able genes into the plant genome instead of causing tumors. The desirable genes become a permanent part of the plant genome, and expression of these genes in plant cells produces desirable products.
One major drawback of the Agrobacterium method is that insertion of bacterial DNA into the plant genome is essentially random. The gene may not be efficiently transcribed at its location, or the insertion of bacterial DNA may knock out an important plant gene by inserting in the middle of it or both may occur. Therefore, the fact that a cell is genetically transformed does not guarantee that it will perform as desired.
Microparticle bombardment is the introduction of foreign DNA constructs into plant cells by attaching the DNA to small metal particles and blasting the particles into plant cells using either a compressed air gun or a gun powered by a 0.22 caliber gun cartridge. This is truly a “brute force” method of introducing DNA into a cell that inadvertently causes many lethal casualties among the bombarded plant cells. However, some plant cells blasted with the DNA-containing metal particles will recover and survive. The plant cells may express the DNA for only a short time (transient expression), because the DNA does not readily integrate into the plant genome, but occasionally the foreign DNA may spontaneously recombine into the plant genome and become permanent.
Other ways of introducing foreign DNA into plant cells include electro oration, microinjection, sonication, and chemical treatment. These methods are not used extensively, because they generally require the production of protoplasts (plant cells that lack their cell walls) from plant cells before transformation. To create protoplasts, the plant cell wall is removed by digestion with the enzymes cellulase and pectinase. Protoplasts are fragile structures, but the absence of a cell wall is desirable because it leaves only the plasma membrane as a barrier to foreign DNA entering a plant cell.
Electroporation uses very brief pulses of high voltage electrical energy to create temporary holes in the plasma membrane through which the foreign DNA can pass. Microinjection involves physically injecting a small amount of DNA into a plant cell using a microscope and an extremely fine needle. Sonication uses ultrasonic waves to punch temporary holes in the plasma membrane, this method is therefore similar to electroporation. Chemical treatment involves the use of polyethylene glycol to render the plasma membrane permeable to foreign DNA.
All the transformation procedures produce only a few transformed cells out of the millions of cells in an explant, so selection of transformed cells is essential.