Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Plant Genetic Engineering: Methodology
Microparticle Bombardment

Microparticle bombardment, or biolistics, is the most promising method for introducing DNA into plant Cells. Spherical gold or tungsten particles, 0.4–1.2 μm in diameter, are coated with DNA precipitated with CaCl₂, spermidine, or polyethylene glycol, and "shot" into cells using a special "gun" powered by gunpowder combustion gases, compressed air, or helium. The particles are accelerated to velocities of 300–600 m/s, penetrating The plant Cell wall and membranes. Their density is such that the cells remain virtually undamaged.

Once inside The Cell, the DNA coating the particles integrates into the plant DNA by some unknown mechanism. The microparticle bombardment method allows for the Transformation of a wide variety of plant species, including monocots and conifers, which cannot be transformed using Agrobacterium.

Microparticle bombardment can also be used to introduce foreign DNA into plant cell Suspensions, cell cultures, meristematic Tissues, immature embryos, protocorms, coleoptiles, and pollen from a wide range of plants (Table 17.3). Furthermore, this method has been used to deliver genes into METABOLISM/14.html">Chloroplasts and Mitochondria. Plasmid DNA dissolved in a buffer can be precipitated onto The surface of the microparticles. This increases transformation frequency by increasing The amount of plasmid DNA; however, it should be noted that excessive amounts of DNA can be lethal to the cell.

Class="center">Table 17.3. Transgenic Plants obtained by microparticle bombardment of various plant cells¹⁾

¹⁾ From Southgate et al., Biotechnol. Adv., 13, 631—651, 1995.

In cells transformed this way, which are identified by the expression of a marker Gene, the introduced DNA is often expressed only transiently. Until the foreign DNA integrates into the plant genome, it is highly likely to be lost during the division of the transformed cells.

Both the integration and expression of foreign genes can depend on the configuration of the vector used for their Introduction. For example, transformation frequency increases when linear rather than Circular DNA is used. Furthermore, during microparticle bombardment, high-molecular-weight Plasmids (>10 kb) can fragment, resulting in lower expression levels of foreign genes compared to smaller plasmids.