integration patterns can be largely controlled by manipulating the configuration of the
introduced DNA (see next section).
10.4.3 The Power and Problems of Direct DNA Introduction
As particle bombardment is a physical method for DNA introduction, complications from
biological interactions with the plant (as withAgrobacterium-mediated transformation) are
avoided. A wide variety of plant tissues can be used as targets for particle bombardment.
These range from embryos, seedlings, shoot apices, leaf disks, microspores, and immature
pollen grains to potato tubers and nodes (Altpeter et al. 2005). Although the foreign DNA
integration patterns (discussed above) can be very complex, this mechanism for DNA
recombination and integration can be an advantage. Various DNAs can be mixed and coin-
troduced using a method calledcotransformation. Reports of 12–15 different DNAs have
been successfully cotransformed into soybean (Hadi et al. 1996) and rice (Chen et al. 1998).
This is potentially useful for pathway engineering, where it is necessary to introduce mul-
tiple genes simultaneously.
Particle bombardment remains the only method that can be used for transformation of
chloroplasts and mitochondria. Plastid transformation (Bock and Khan 2004) is useful in
cases where large amounts of the transgene product are needed. The integration of
foreign DNA into plastid DNA is also simple because integration events are less
complex compared to nuclear transformation. For plastid transformation, the foreign
DNA is precisely constructed so that it combines with similar sequences in the target
plastid DNA, usinghomologous recombination. Another advantage of plastid transform-
ation is that transgene escape via pollen is avoided since plastids are only maternally inher-
ited in most plant species. However, like the floral dip method, this technique is currently
limited to a small number of species.
In hand with the numerous merits of particle bombardment, there are certain drawbacks
that limit the use of particle bombardment. The main perceived limitations are the random-
ness of DNA integration and the high copy number of introduced DNAs. As with most
methods of DNA introduction, the position and orientation of the transgene in the plant
chromosome will differ with every transformation event. The location of the transgene
within the target chromosome will influence the expression of that gene. Transgenes in
more active regions of genomic DNA will express at higher levels, while integration in
less active areas will lead to lower expression:position effects. More importantly, the
number of copies of introduced DNA can be incredibly high, leading to inactivity of the
introduced DNAs (Taylor and Fauquet 2002). One might think that the presence of
many copies of a particular transgene would result in very high expression. But expression
of the transgene is often downregulated by the plant, a phenomenon known ascosuppres-
sion,homology-dependent silencing,RNA interference(RNAi), orRNA silencing(Zhong
2001; Butaye et al. 2005). Selection of plant cells or tissue showing uniform transgene
expression is critical. Several techniques have been developed to minimize variation in
transgene expression from particle bombardment. These methods are similarly applicable
to other direct DNA introduction methods (discussed later in this chapter).
10.4.4 Improvements in Transgene Expression
Variation in transgene expression resulting from particle bombardment can be reduced to
some extent by modifying the introduced DNA. Since high-copy-number integration
10.4. PARTICLE BOMBARDMENT 259