The Biolistic Transformation Method
The biolistic transformation method was first described in 1987 by John Sanford and colleagues at Cornell University (USA) who used DNA-coated tungsten particles to literally shoot them into onion cells (Sanford et al., 1987).
 |
| Fig. 221 : Particle gun for biolistic gene transfer (Photograph courtesy of A. Milligan, ACPFG, Australia) |
The principle of direct gene transfer using the particle gun (Fig. 221) is still the same today, although gunpowder has been replaced by gas pressure and the chemically inert metal gold is used instead of tungsten.
Technically, the gun works as follows: helium gas flows into the pressure chamber, where pressure builds up behind a plastic disc of defined strength, the rupture disc.
When the gas pressure in the top chamber reaches a threshold value, the rupture disc bursts and releases the pressure downwards into the vacuum chamber.
The pressure accelerates the DNA-coated gold particles that reside close to the burst rupture disc until they hit the cells of the explants, the immature embryos on a Petri dish containing medium.
The microprojectiles have to penetrate the walls of the plant cells so that the DNA can get into solution in the aqueous cell content. Only a small proportion of the cells that have been hit will integrate the DNA into their genome.
In most cells the DNA is degraded by enzymes or lost during subsequent cell divisions. In these latter cases the transformation is called "transient" in contrast to the desired "stable" transformation.
The first stable transformations of cereal plants using immature embryos were achieved at the beginning of the 1990 s.
Shortly after the success with rice in 1991 (Christou et al., 1991) wheat followed in 1993 (Vasil et al., 1993; Weeks et al., 1993; Becker et al., 1994).
The initially low success rate for generating transgenic wheat plants of about 1% (one transgenic plant regenerated out of 100 isolated and bombarded immature embryo explants) has since increased dramatically and may be as much as 60% with certain cultivars, depending on the laboratory reporting.
The biolistic transformation method makes use of physical processes to transfer the gene into the plant cells. This makes it a versatile and effective transformation method that is not limited to a cell type, species or genotype.
Discussed disadvantages of the biolistic method are a tendency to integrate multiple copies of the transgene into the genome together with observed fragmentation of transgene copies, especially when large DNA fragments are transferred.
Multiple integrations of the same sequence can cause inhibition of gene expression by the plant so that the trait encoded by the transgene is not displayed.
An alternative to the biolistic transformation method, Agrobacterium tumefaciens-mediated transformation, is now being used for genetic transformation of a wide range of species.
Post a Comment