PARTICLE BOMBARDMENT TECHNOLOGY
Another strategy for getting
a gene of interest into plant tissue is to blast the DNA through the plant cell
walls with a particle gun. Unlike Ti plasmid transfer by Agrobacterium, this
technique works with all types of plants. The basic idea is that DNA is carried
on microscopic metal particles. These are fired by a gun into plant tissue and
penetrate the plant cell walls. This technique is rather nonspecific, yet it
has been very successful in the plant world.
First, either a leaf disk
(just a round piece of leaf tissue) or a piece of callus is isolated from the
plant, placed on a dish, and put in a vacuum chamber. The DNA to be inserted
(carrying the gene of interest, with any promoter and enhancer elements, plus
selectable markers) is coated on microscopic gold or tungsten beads. Gold beads
are preferable because tungsten can be toxic to some plants.
The beads are placed at the
end of a plastic bullet. One variant of the method uses a blast of air or
helium to project the bullet toward the sample. In the first gene guns, actual
firearm blanks were used to accelerate the bullet. Between the bullet and plant
tissue is a plastic meshwork stop. When the bullet hits the stop, the
DNA-coated beads are thrown forward through the meshwork and continue on
through the vacuum chamber and into the plant tissue. An alternative method is
to accelerate the beads by a strong electrical discharge. The high voltage
vaporizes a water droplet, and the resulting shock wave propels a thin metal
sheet covered with the particles at a mesh screen. The screen blocks the metal
sheet but allows the DNA-coated particles to accelerate through into the plant
tissue (Fig. 14.7). One advantage to this method is that the strength of the
electrical discharge can be controlled; therefore, the amount of penetration
into the tissue can be changed at will.
When the beads penetrate the
tissue, some will actually enter the cytoplasm or nucleus of the leaf or callus
cells. The DNA dissolves off the beads inside the cells and the DNA is free to
recombine with the chromosomal DNA of the plant (Fig. 14.8). The leaf or callus
tissue is then transferred to selection media where the cells that integrated
the DNA carrying the selectable marker are able to grow, but other cells die.
The transformed plants are regenerated using tissue culture techniques, and
finally screened for the gene of interest.
Particle guns have also been
used on animal tissues. In addition, scientists have modified the technology in
order to transform DNA into the mitochondria of yeast, as well as the
chloroplasts of Chlamydomonas, a
small green alga.